Irritable bowel syndrome and active inflammatory bowel disease diagnosed by faecal gas analysis.

BACKGROUND: Inflammatory bowel disease and irritable bowel syndrome may present in a similar manner. Measuring faecal calprotectin concentration is often recommended to rule out inflammatory bowel disease, however, there are no tests to positively diagnose irritable bowel syndrome and invasive tests are still used to rule out other pathologies. AIM: To investigate a platform technology for diagnosing inflammatory bowel disease and irritable bowel syndrome based on faecal gas. METHODS: The platform technology is composed of a gas chromatography column coupled to a metal oxide gas sensor (OdoReader) and a computer algorithm. The OdoReader separates the volatile compounds from faecal gas and the computer algorithm identifies resistance patterns associated with specific medical conditions and builds classification models. This platform was applied to faecal samples from 152 patients: 33 patients with active inflammatory bowel disease; 50 patients with inactive inflammatory bowel disease; 28 patients with irritable bowel syndrome and 41 healthy donors (Control). RESULTS: The platform classified samples with accuracies from 75% to 100% using rigorous validation schemes: namely leave-one-out cross-validation, 10-fold cross-validation, double cross-validation and their Monte Carlo variations. The most clinically important findings, after double cross-validation, were the accuracy of active Crohn's disease vs. irritable bowel syndrome (87%; CI 84-89%) and irritable bowel syndrome vs. controls (78%; CI 76-80%). These schemes provide an estimate of out-of-sample predictive accuracy for similar populations. CONCLUSIONS: This is the first description of an investigation for the positive diagnosis of irritable bowel syndrome, and for diagnosing inflammatory bowel disease.

The use of a gas chromatograph coupled to a metal oxide sensor for rapid assessment of stool samples from irritable bowel syndrome and inflammatory bowel disease patients.

There is much clinical interest in the development of a low-cost and reliable test for diagnosing inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS), two very distinct diseases that can present with similar symptoms. The assessment of stool samples for the diagnosis of gastro-intestinal diseases is in principle an ideal non-invasive testing method. This paper presents an approach to stool analysis using headspace gas chromatography and a single metal oxide sensor coupled to artificial neural network software. Currently, the system is able to distinguish samples from patients with IBS from patients with IBD with a sensitivity and specificity of 76% and 88% respectively, with an overall mean predictive accuracy of 76%.

A review of the volatiles from the healthy human body.

A compendium of all the volatile organic compounds (VOCs) emanating from the human body (the volatolome) is for the first time reported. 1840 VOCs have been assigned from breath (872), saliva (359), blood (154), milk (256), skin secretions (532) urine (279), and faeces (381) in apparently healthy individuals. Compounds were assigned CAS registry numbers and named according to a common convention where possible. The compounds have been grouped into tables according to their chemical class or functionality to permit easy comparison. Some clear differences are observed, for instance, a lack of esters in urine with a high number in faeces. Careful use of the database is needed. The numbers may not be a true reflection of the actual VOCs present from each bodily excretion. The lack of a compound could be due to the techniques used or reflect the intensity of effort e.g. there are few publications on VOCs from blood compared to a large number on VOCs in breath. The large number of volatiles reported from skin is partly due to the methodologies used, e.g. collecting excretions on glass beads and then heating to desorb VOCs. All compounds have been included as reported (unless there was a clear discrepancy between name and chemical structure), but there may be some mistaken assignations arising from the original publications, particularly for isomers. It is the authors' intention that this database will not only be a useful database of VOCs listed in the literature, but will stimulate further study of VOCs from healthy individuals. Establishing a list of volatiles emanating from healthy individuals and increased understanding of VOC metabolic pathways is an important step for differentiating between diseases using VOCs.

Towards point of care testing for C. difficile infection by volatile profiling, using the combination of a short multi-capillary gas chromatography column with metal oxide sensor detection.

Rapid volatile profiling of stool sample headspace was achieved using a combination of short multi-capillary chromatography column (SMCC), highly sensitive heated metal oxide semiconductor (MOS) sensor and artificial neural network (ANN) software. For direct analysis of biological samples this prototype offers alternatives to conventional GC detectors and electronic nose technology. The performance was compared to an identical instrument incorporating a long single capillary column (LSCC). The ability of the prototypes to separate complex mixtures was assessed using gas standards and homogenised in house 'standard' stool samples, with both capable of detecting more than 24 peaks per sample. The elution time was considerably faster with the SMCC resulting in a run time of 10 minutes compared to 30 minutes for the LSCC. The diagnostic potential of the prototypes was assessed using 50 C. difficile positive and 50 negative samples. The prototypes demonstrated similar capability of discriminating between positive and negative samples with sensitivity and specificity of 85% and 80% respectively. C. difficile is an important cause of hospital acquired diarrhoea, with significant morbidity and mortality around the world. A device capable of rapidly diagnosing the disease at the point of care would reduce cases, deaths and financial burden.

Volatiles from oral anaerobes confounding breath biomarker discovery.

The levels of compounds in exhaled mouth air do not necessarily reflect levels in the systemic circulation as bacteria can bio-transform substrates to produce compounds within the mouth. This should be of concern to researchers measuring breath volatiles with the aim of diagnosing systemic or metabolic conditions as very little is known about the origin of many compounds detected on the breath. This pilot study investigated the production of volatile compounds by bacterial communities present within the mouth. Solid-phase micro-extraction was used to extract volatiles from the headspace gas of two Gram-positive and two Gram-negative bacterial cultures known to be present within the mouth and from tongue biofilm microflora. Analyses were undertaken using gas chromatography mass spectrometry. Between 64 and 82 volatile compounds were detected from sampling the headspace gas above each of the cultures. Gram-negative anaerobes were found to produce more volatile sulfur compounds (VSCs) and amines. Solobacterium moorei, a Gram-positive species was however found to produce higher levels of acids, hydrocarbons, alcohols and aldehydes than the other species studied. Tongue-scrape biofilm systems at lower pH gave more hydrocarbons, ketones and fatty acids whilst at higher pH more alcohols, aldehydes, VSCs and amines were detected in the headspace. The results show that a number of compounds detected in mouth breath are produced by anaerobic bacteria in tongue biofilms. Thus, the contribution of volatiles from oral anaerobes cannot be ignored and more research is required to identify the major source of breath compounds as this will help determine their clinical significance as indicators of systemic disease or metabolic disorders in the body.

The importance of methane breath testing: a review.

Sugar malabsorption in the bowel can lead to bloating, cramps, diarrhea and other symptoms of irritable bowel syndrome as well as affecting absorption of other nutrients. The hydrogen breath test is now a well established noninvasive test for assessing malabsorption of sugars in the small intestine. However, there are patients who can suffer from the same spectrum of malabsorption issues but who produce little or no hydrogen, instead producing relatively large amounts of methane. These patients will avoid detection with the traditional breath test for malabsorption based on hydrogen detection. Likewise the hydrogen breath test is an established method for small intestinal bacterial overgrowth (SIBO) diagnoses. Therefore, a number of false negatives would be expected for patients who solely produce methane. Usually patients produce either hydrogen or methane, and only rarely there are significant co-producers, as typically the methane is produced at the expense of hydrogen by microbial conversion of carbon dioxide. Various studies show that methanogens occur in about a third of all adult humans; therefore, there is significant potential for malabsorbers to remain undiagnosed if a simple hydrogen breath test is used. As an example, the hydrogen-based lactose malabsorption test is considered to result in about 5-15% false negatives mainly due to methane production. Until recently methane measurements were more in the domain of research laboratories, unlike hydrogen analyses which can now be undertaken at a relatively low cost mainly due to the invention of reliable electrochemical hydrogen sensors. More recently, simpler lower cost instrumentation has become commercially available which can directly measure both hydrogen and methane simultaneously on human breath. This makes more widespread clinical testing a realistic possibility. The production of small amounts of hydrogen and/or methane does not normally produce symptoms, whereas the production of higher levels can lead to a wide range of symptoms ranging from functional disorders of the bowel to low level depression. It is possible that excess methane levels may have more health consequences than excess hydrogen levels. This review describes the health consequences of methane production in humans and animals including a summary of the state of the art in detection methods. In conclusion, the combined measurement of hydrogen and methane should offer considerable improvement in the diagnosis of malabsorption syndromes and SIBO when compared with a single hydrogen breath test.

A pilot study of faecal volatile organic compounds in faeces from cholera patients in Bangladesh to determine their utility in disease diagnosis.

The aim of this pilot study was to analyse the volatile organic compounds in faecal samples collected from cholera patients in Bangladesh to determine biomarkers that could be used for disease diagnosis. Samples were collected from patients at the International Centre for Diarrhoeal Disease Research, Dhaka, Bangladesh and also from healthy controls at the same institution. The volatile organic compounds were extracted from the headspace above the sample using solid phase microextraction and analysed using gas chromatography-mass spectrometry. A biomarker was identified in the cholera samples that could be used for disease diagnosis.

A sensor system for monitoring the simple gases hydrogen, carbon monoxide, hydrogen sulfide, ammonia and ethanol in exhaled breath.

A sensor array system was constructed incorporating electrochemical sensors for hydrogen, carbon monoxide, hydrogen sulfide and ethanol, a ceramic sensor for total volatiles and a dye-based optical ammonia sensor. The system was calibrated using standard gases balanced with dry air. Limit of detection and % relative standard deviation values (n = 10) for the sensors in the array are hydrogen (0.1 ppm, 2.6%), carbon monoxide (0.4 ppm, 2.1%), ethanol (0.5 ppm, 1.5%), hydrogen sulfide (0.1 ppm, 1.5%) and ammonia (0.6 ppm, 10.7%). Humidity effects were assessed by calibrating with humidified standard gases (hydrogen, carbon monoxide) or spiked breath samples in Tedlar bags (hydrogen sulfide, ethanol and ammonia). The calibration data were used to establish a cross-sensitivity matrix. The concentration of breath volatiles was found to be dependent on exhalation rate and exhalation volume. A test protocol based on these data required volunteers to exhale 1 litre of breath at a rate between 7.5 and 17.5 l min(-1). Sensor responses were measured for 40 s then purged at 7 l min(-1) (150 s). A longitudinal study was undertaken of ten asymptomatic volunteers over a five-day period. Volunteers ate an ad hoc diet, but fasted prior to giving the first breath sample and then gave samples every hour for 8 h. Breath hydrogen levels for volunteers showed large variations within a day and also from day to day. Fasting levels ranged between 0.3 and 34.1 ppm (mean 9.1 ppm). The carbon monoxide levels for non-smokers were between 0.6 and 4.9 ppm (mean 2.1 ppm), whilst for smokers they were between 8.3 and 18.7 ppm (mean 12.8 ppm). The measured levels of other gases on breath were as follows: hydrogen sulfide (0-1.3 ppm, mean 0.33 ppm), ethanol (0-3.9 ppm, mean 0.62 ppm) and ammonia (0-1.3 ppm mean 0.42 ppm). The system was capable of direct quantitative measurements of low concentrations of a range of volatiles on exhaled breath. The measured values for compounds on the breath of asymptomatic volunteers were in broad agreement with quoted literature ranges. The system will now be assessed in a clinical setting.

The characteristics of novel low-cost sensors for volatile biomarker detection.

For breath analyses, volatile detectors capable of sensing extremely low concentrations in the sub-ppm range are required. Novel room temperature sensors were fabricated based on ultraviolet light activation of nanoparticulate metal oxide surfaces using light emitting diodes. These sensors gave reversible electrical resistance changes in the low ppm/ppb range to volatile organic compounds found in breath, including acetone, acetaldehyde, pentane and ethanol.

A comparative study of the analysis of human urine headspace using gas chromatography-mass spectrometry.

First-void urine samples were obtained from 24 elderly, asymptomatic men (median age 62.9 years). The headspace above pH adjusted urine samples were extracted using a carboxen/polydimethylsiloxane solid phase micro-extraction fibre and the volatile organic compounds analysed by gas chromatography/mass spectrometry. A total of 147 compounds were identified in the headspace of urine. The acidified samples recorded a total of 92 compounds, 27 of which were ubiquitous, basified samples 70 compounds, with 12 ubiquitous and unmodified pH samples 49, with 6 ubiquitous. Five compounds were ubiquitous irrespective of pH: acetone, methylene chloride, 4-heptanone, 2-pentanone and 2-butanone. A comparative analysis of unfrozen and frozen-thawed urine (stored at room temperature for 0, 1 and 8 h) showed that samples retained the same number of compounds although variations in the peak areas for some compounds were observed.

An analysis of volatiles in the headspace of the faeces of neonates.

A gas chromatography/mass spectrometry (GCMS) analysis of the headspace from the faeces of neonates was undertaken to record the volatiles associated with preterm babies on a neonatal unit. The compounds ethanol, acetone, 2-ethyl-1-hexanol, 3-methylbutanal, hexanal and 2,3-butanedione occurred with the highest frequency. The volatiles analysed were then compared to a previously published study of the volatiles from asymptomatic adult faeces. Fewer compounds were found in the neonatal faeces and virtually no sulfides were detected, in contrast to the adult samples where carbon disulfide, dimethyl disulfide and dimethyl sulfide were ubiquitous. In addition, 7 of the most abundant 15 volatile compounds were found to be aldehydes, while in contrast only 2, acetaldehyde and benzaldehyde, were present in the most abundant 15 compounds found in the headspace of adult faeces. 2-Ethyl-1-hexanol was considerably more abundant in the neonate stool compared to adult stool, and probably reflects high exposure to plastic materials containing plasticizers. The potential of disease diagnoses from the analysis of volatiles emitted from neonate faeces is discussed.

The rapid detection of methyl tert-butyl ether (MtBE) in water using a prototype gas sensor system.

The gasoline additive Methyl-tertiary-Butyl Ether (MtBE) is the second most common contaminant of groundwater in the USA and represents an important soil contaminant. This compound has been detected in the groundwater in at least 27 states as a result of leaking underground storage facilities (gasoline storage tanks and pipelines). Since the health effects of MtBE are unclear the potential threat to drinking water supplies is serious. Therefore, the ability to detect MtBE at low levels (ppb) and on-line at high-risk groundwater sites would be highly desirable. This paper reports the use of 'commercial' and metal oxide sensor arrays for the detection of MtBE in drinking and surface waters at low ppb level (microg.L(-1) range). The output responses from some of the sensors were found to correlate well with MtBE concentrations under laboratory conditions.

A novel method for rapidly diagnosing the causes of diarrhoea.

BACKGROUND: The microbiological diagnosis of infectious diarrhoea may take several days using conventional techniques. In order to determine whether flatus can be used to make a rapid diagnosis, the volatile organic compounds associated with diarrhoea were analysed. METHODS: Stool samples were collected from 35 patients with infectious diarrhoea and from six healthy controls. Gaseous compounds were extracted from a headspace using solid phase microextraction and analysed using gas chromatography and mass spectroscopy. RESULTS: Characteristic patterns of volatile gases were found for the main causes of infectious diarrhoea in hospitals. Furan species without indoles indicated Clostridium difficile, ethyl dodecanoate indicated rotavirus, ammonia without ethyl dodecanoate suggested other enteric viruses, and the absence of hydrocarbons and terpenes indicated Campylobacter infection. CONCLUSION: These results could be the basis of rapid near patient diagnosis of infectious diarrhoea.

Development of an electrochemical assay for 2,6-dinitrotoluene, based on a screen-printed carbon electrode, and its potential application in bioanalysis, occupational and public health.

Screen-printed carbon electrodes (SPCEs) have been successfully exploited as disposable sensors for the measurement of 2,6-dinitrotoluene (2,6-DNT) using a stripping voltammetric method. Initial investigations were undertaken using cyclic voltammetry (CV) to characterise the redox behaviour at the SPCEs. Further studies were then performed to deduce the optimum applied potential and accumulation time for the stripping voltammetric procedure. In addition, a study was carried out to ascertain whether small volumes of samples could be reliably used for analysis. From these studies it was shown that a 100 microl aliquot of sample could be analysed and the calibration plot was linear from 161 ng ml(-1) to 137 microg ml(-1) (R(2)=0.9991), the former concentration being the detection limit. The effects of the major components of human saliva at concentrations normally present were investigated. Of the individual components tested, only Cl(-) and albumen were found to interfere. The presence of the latter could be easily overcome by the addition of (NH(4))(2)SO(4). An interference study was also carried out on some inorganic and organic species that may be present in water samples. The sensors were evaluated by carrying out 2,6-DNT determinations on spiked and unspiked human saliva, dust wipe and potable water samples. Mean recoveries of 47.5, 73.4 and 102.4% were obtained; coefficients of variation of 7.88, 6.63 and 6.42% were calculated for a concentration of 9.1 microg ml(-1) in water, 10.6 microg ml(-1) saliva samples, and 141.1 ng cm(-2) for dust wipe samples, respectively. The performance characteristics show that the method holds promise and reliable data may be obtained for 2,6-DNT in bioanalysis and public health.

Ammonia vapour in the mouth as a diagnostic marker for Helicobacter pylori infection: preliminary 'proof of principle' pharmacological investigations.

Most current non-invasive tests for Helicobacter pylori depend on the conversion of labelled (13C or 14C) urea to labelled carbon dioxide (13CO2 or 14CO2) and ammonium (NH4+) by the enzyme urease, with the labelled CO2 detected in exhaled air. Despite suggestions going back over a number of years, the alternative possibility of using NH4+ (in the form of gaseous ammonia [NH3]) as the test parameter has received little or no attention. However, this approach is now being explored using a chemiresistive sensor detecting sub-parts per million concentrations of NH3. An in vitro 'glass stomach' (containing various volumes of hydrochloric acid [HCl] and ammonium chloride [NH4Cl]) was used to evaluate the means of increasing 'gastric' pH to that of the NH4+-->NH3 transition that occurs significantly at pH 9.24. This 'stomach' also was used to study mechanisms by which NH3 may be expelled in a pulse (as a surrogate belch), either by the in situ production of CO2 or through an exogenous source. On the basis of the protocols developed, H. pylori-negative subjects were tested before and after ingestion of 10 mg NH4Cl (as a surrogate for bacteria-produced NH4,), and H. pylori-positive subjects were tested without taking urea or NH4Cl. 'Intragastric' pH in the in vitro 'glass stomach' could be increased above pH 9.24 by adding a mixture of 15-30 mL magnesium hydroxide mixture (or the proprietary equivalent) and 50 mL water, and the resulting NH3 expelled by adding 100 mL CO2-saturated cold water (sparkling water). In vivo, NH3 levels in the oral cavity of H. pylori-negative subjects were increased after ingestion of 10 mg NH4Cl; however, levels in the oral cavity of a small number of H. pylori-positive subjects were two- to threefold higher after magnesium hydroxide and sparkling water. On the basis of in vitro studies, an in vivo protocol was developed to increase gastric pH above that required for the NH4+-->NH3 transition, and a mechanism established to release the NH3 into the oral cavity. Preliminary in vivo data confirm the chemiresistive sensor is sufficiently sensitive to NH3 to distinguish H. pylori-negative subjects who have taken 10 mg NH4Cl from those who have not, and clearly distinguish H. pylori-negative subjects from H. pylori-positive subjects. Ingestion of urea or other labelled tracers is not required, nor is belching; and the sensor takes less than two minutes to reach a maximum response. The data provide good evidence that the chemiresistive detection of NH3 has considerable potential as a rapid, point-of-care diagnostic test for H. pylori infection.

Identifying bacteria in human urine: current practice and the potential for rapid, near-patient diagnosis by sensing volatile organic compounds.

Urinary tract infection (UTI) represents a significant burden for the National Health Service. Extensive research has been directed towards rapid detection of UTI in the last thirty years. A wide range of microbiological and chemical techniques are now available to identify and quantify bacteria in urine. However, there is a clear and present need for near, rapid, sensitive, reliable analytical methods, preferably with low-running costs, that could allow early detection of UTI and other diseases in urine. Here we review the "state of the art" of current practice for the detection of bacteria in urine and describe the advantages of the recent "e-nose" technology as a potential tool for rapid, near-patient diagnosis of UTI, by sensing volatile organic compounds (VOCs).