WO2015177523A1 - Detection of bacterial infection - Google Patents

Detection of bacterial infection Download PDF

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Publication number
WO2015177523A1
WO2015177523A1 PCT/GB2015/051455 GB2015051455W WO2015177523A1 WO 2015177523 A1 WO2015177523 A1 WO 2015177523A1 GB 2015051455 W GB2015051455 W GB 2015051455W WO 2015177523 A1 WO2015177523 A1 WO 2015177523A1
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WIPO (PCT)
Prior art keywords
gas
chamber
faecal sample
sample
outlet
Prior art date
Application number
PCT/GB2015/051455
Other languages
French (fr)
Inventor
Mitesh Patel
Christopher Walton
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Cranfield University
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Publication of WO2015177523A1 publication Critical patent/WO2015177523A1/en

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/4022—Concentrating samples by thermal techniques; Phase changes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/0004—Gaseous mixtures, e.g. polluted air
    • G01N33/0009—General constructional details of gas analysers, e.g. portable test equipment
    • G01N33/0011—Sample conditioning
    • G01N33/0021—Sample conditioning involving the use of a carrier gas for transport to the sensor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/0004—Gaseous mixtures, e.g. polluted air
    • G01N33/0009—General constructional details of gas analysers, e.g. portable test equipment
    • G01N33/0027—General constructional details of gas analysers, e.g. portable test equipment concerning the detector
    • G01N33/0036—General constructional details of gas analysers, e.g. portable test equipment concerning the detector specially adapted to detect a particular component
    • G01N33/0047—Organic compounds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/48707—Physical analysis of biological material of liquid biological material by electrical means
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/02—Devices for withdrawing samples
    • G01N1/22—Devices for withdrawing samples in the gaseous state
    • G01N1/2226—Sampling from a closed space, e.g. food package, head space
    • G01N2001/2229—Headspace sampling, i.e. vapour over liquid
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/33—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Clostridium (G)

Definitions

  • the present invention relates to detection of bacterial infections, in particular infections of
  • Clostridium difficile (C. difficile) in the faeces of humans and animals.
  • GDH glutamate dehydrogenase
  • test kits have limited shelf life (expiration date) and need to be stored between 2-8°C, although they must be at room temperature at time of use. Tests cannot be reused. Faecal specimens for testing should ideally be no older than 24 hours, but can be tested up to 72 hours following collection. Frozen samples may lose activity due to the freeze-thaw cycle and degradation of toxins. Such frozen samples must also be thawed at room temperature.
  • GCMS gas chromatography-mass spectrometry
  • the present inventors have previously disclosed the development of a device for the diagnosis of C. Difficile Associated Diarrhoea employing the detection in faecal headspace of volatile organic compounds produced by C. Difficile and based on quantitative analysis to determine the concentrations of p-cresol produced in faecal samples from infected and non- infected patients.
  • a method of analysing a faecal sample comprising the steps of:
  • the method may comprise the step of continuously feeding volumes of gas into the chamber and simultaneously continuously exhausting volumes of gas from the chamber, i.e. there may be a continuous flow of gas through the chamber.
  • the continuous flow of gas may be at a rate of about 100ml per minute.
  • the method may comprise the step of analysing the continuously exhausted volumes of gas, i.e. continually analysing the flow of gas exhausted from the chamber.
  • the method may comprise the step of incubating the faecal sample prior to feeding gas into the chamber.
  • the method may comprise the step of incubating the sample at about 40 ° C.
  • the method may comprise the step of incubating the sample for about 5 minutes prior to feeding gas into the chamber.
  • the method may comprise the step of sucking gas out of the chamber.
  • the method may comprise the step of sucking ambient air in to the chamber.
  • the ambient air may be filtered prior to entry into the chamber.
  • the ambient air may be filtered to remove hydrocarbons.
  • the outlet may be fluidly connectable to an analyser.
  • the outlet may also be fluidly connectable to an analyser bypass path.
  • Analyser and analyser bypass path may both be fluidly connected to a suction pump.
  • the method may comprise the step of switching the fluid connection of the outlet between the analyser and the bypass path without interrupting the continuous flow of gas through the chamber.
  • the method may comprise the step of generating a signal indicative of the presence of the Clostridium difficile bacterium in the faecal sample in dependence on the concentration indicated by the analysis of one or more compounds selected from the group of 1-Propanol, Butanal 3 -methyl, Propanoic acid ethyl ester, Isovaleric acid, 4-Heptanone, Phenol 4-methyl, Indole and Hexanoic acid, in particular Phenol 4-methyl (p-cresol).
  • the method may comprise the step of generating a signal indicative of the presence of the Clostridium difficile bacterium in the faecal sample where the concentration indicated by the analysis of the one or more compounds selected from the group of 1-Propanol, Butanal 3 -methyl, Propanoic acid ethyl ester, Isovaleric acid, 4-Heptanone, Phenol 4-methyl and Indole exceeds a predetermined threshold for that compound.
  • the method may comprise the step of generating a signal indicative of the presence of the Clostridium difficile bacterium in the faecal sample where the concentration of Hexanoic acid indicated by the analysis is lower than a predetermined threshold.
  • a faecal sample chamber having an inlet and an outlet
  • a pump configured to feed a volume of gas through the inlet, into contact with a faecal sample and to exhaust said volume of gas through the outlet;
  • an analyser for analysing the chemical composition of said volume of gas.
  • Figure 1 is a schematic illustrating an embodiment of the present invention
  • Figures 2-8 and 9 are box and whisker plots for 'presence' and 'absence' compounds respectively.
  • Figure 10 is a flow chart illustrating the method according to the invention.
  • the apparatus comprises a chamber 20 defined by a container 28 - in this case a 100ml DuranTM flask - and sealed except for a gas inlet 22 and a gas outlet 24 formed in a seal 26.
  • Seal 26 can be temporarily broken to allow a cup 21 - typically of 2cm diameter - filled with a faecal sample S to be placed on the floor 30 of the chamber. Filling the cup 21 to the brim (and scraping the surface of the sample with a scraper ('doctor blade') as necessary to remove any excess above the brim) ensures a consistent, known sample area A.
  • Container 28 slots into an insulated heating jacket 36 comprising an aluminium mesh 38 surrounded by one or more heating elements 40 which are in turn surrounded by insulation 42.
  • controller 44 controls the electricity supply to the elements in dependence on the signal from a temperature sensor 46 located at the base of the jacket, adjacent the base of the container and the floor of the chamber. In the example shown, the temperature adjacent the base of the container is maintained at approximately 40°C.
  • a pump 34 is started. As indicated by arrows, pump 34 sucks ambient air A through a hydrocarbon trap 32 and gas inlet 22 into the chamber 20 where it contacts the faecal sample S and is then exhausted through gas outlet 24. Trap 32 removes hydrocarbon-containing contaminants which would otherwise interfere with subsequent analysis. Pump 34 provides a continuous, constant flow of 100 ml min "1 at all times so as to allow the sample to reach equilibrium. Such an arrangement also allows data on the rate of emission from the sample, e.g. in milligrams per area A of sample per unit of time, to be collected as necessary.
  • the exhausted gas is drawn via valves Vi and V 2 either through an analyser 48 or through a bypass tube 50 and thence to the pump 34 where it is exhausted to atmosphere as indicated at Z.
  • analyser 48 is a sampling thermal desorption (TD) tube. Gas is allowed to flow through the tube for a period (in the example shown, five minutes) following which, by operation of valves Vi and V 2 , the flow is switched to a bypass tube 50 as indicated by arrows 52.
  • Bypass tube may have the same flow resistance characteristics as the TD tube so as not to upset the flow through the chamber. Switching allows the sampling thermal desorption tube 48 to be removed for analysis. A fresh sampling tube can be inserted and/or the sample can be removed from the chamber and a fresh sample can be analysed and/or a new container can be used, as appropriate.
  • analysis of the TD tube is as follows.
  • An internal standard solution comprising 50ng d8-toluene (Supelco Cat No. 48593) in methanol is added to each tube according to the manufacturer's instructions (Markes International Ltd, Llantrisant, UK).
  • TD-GC-MS thermal desorption gas chromatography mass spectrometry
  • the analytical instrumentation comprising a Series 2 Unity thermal desorber with Series 2 Ultra autosampler connected to an Agilent model 7890 gas chromatograph and ALMSCO Bench-ToF time-of-flight mass spectrometer (Markes International, Llantrisant, UK), the gas chromatograph being fitted with a DB5 column of dimensions 60m x 0.4mm x 0.25mm (Agilent Technologies UK Limited, Stockport, UK).
  • the carrier gas is CP grade helium (BOC gases, Guildford, UK) passed through a combined trap for removal of hydrocarbons, oxygen and water vapour.
  • TD tubes are initially purged for 1 minute in order to remove air and water vapour then desorbed at 300° C for 8 minutes onto a secondary cold trap (Materials emission trap,
  • the MS is operated from 20 - 450 amu at an effective scan rate of approximately 2.2 Hz.
  • Compound identification is achieved using Chemstation (enhanced data analysis) software and the National Institute of Standards and Technology (NIST) mass spectral library. Quantification is achieved by comparing the area of each compound peak with the peak area associated with the known amount of d8-toluene.
  • the invention may be used to identify the presence or absence of the Clostridium difficile bacterium in a faecal sample. Specifically, it has been discovered that there is a statistically significant correlation between the presence of Clostridium difficile bacterium in a faecal sample and the concentration above a certain level in the gas analysed according to the invention of one or more of the compounds 1-Propanol, Butanal 3 -methyl, Propanoic acid ethyl ester, Isovaleric acid, 4-Heptanone, Phenol 4-methyl and Indole. It has also been discovered that there is a statistically significant correlation between the presence of Clostridium difficile bacterium in a faecal sample and the concentration below a certain level in the gas analysed according to the invention of Hexanoic acid.
  • the correlations are illustrated in the box and whisker plots of figures 2-8 (for the first seven 'presence' compounds) and figure 9 (for the eighth 'absence' compound).
  • the plots each show a difference, significant at the 5% level of probability, in median concentration of a compound between a group (I) of clinical samples known to be negative for C Diffi; and a group (II) of clinical samples known to be positive for C Diffi.
  • group (III) of fresh controls from healthy volunteers
  • a group (IV) of frozen controls from healthy volunteers).
  • Phenol 4 methyl- is particularly advantageous as there is (a) a large difference of around 7.5000 in median concentration between group I and group II, and (b) no overlap between the boxes defining the upper and lower quartiles of group I and group II.
  • ROC characteristic indicates the overall performance of a compound in distinguishing between infected and non- infected samples, an ideal test having an area of 1 (or zero for an absence marker).
  • the presence markers according to the present invention have a lower bound (95% confidence interval) greater than or equal to 0.6 while the absence marker according to the present invention has an upper bound less than or equal to 0.4.
  • the Chemical Abstract Service (CAS) identification number for each compound is also indicated.
  • the present invention can be used to identify the presence of the Clostridium difficile bacterium in a faecal sample as illustrated in figure 10.
  • the sample is placed in the chamber, incubated and subject to gas flow as discussed above.
  • the exhausted gas is then analysed to establish values of concentration of 1-Propanol, Butanal 3 -methyl, Propanoic acid ethyl ester, Isovaleric acid, 4-Heptanone, Phenol 4-methyl and Indole. These values are then compared with predetermined thresholds for each compound and, where a value exceeds a threshold, a signal is generated indicative of the presence of C.Diff in the sample.
  • the exhausted gas is also analysed to establish a value of concentration of Hexanoic acid and, where a value is lower than a predetermined threshold, a signal is generated indicative of the presence of C.Diff in the sample.

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Abstract

A method of analysing a faecal sample comprises the steps of providing a sealed chamber (20) having an inlet (22) and an outlet (24); placing a faecal sample (S) in the chamber; thereafter feeding a volume of gas through the inlet and into contact with the faecal sample; thereafter exhausting said volume of gas through the outlet; and there after analysing the chemical composition of said volume of gas.

Description

TITLE: DETECTION OF BACTERIAL INFECTION
TECHNICAL FIELD
The present invention relates to detection of bacterial infections, in particular infections of
Clostridium difficile (C. difficile) in the faeces of humans and animals.
BACKGROUND ART
A common method of C. difficile diagnosis is by the glutamate dehydrogenase (GDH) enzyme immunoassay. This detects for the GDH antigen and the toxins A and B in a single reaction well. More of a test to confirm the absence of C. difficile, the test is non-specific and needs to be confirmed by cell culture which can take 48 hours and may require a specialist laboratory. The test requires the use of reagents, diluent (protein solution), wash buffer, substrate containing trimethylbenzine and conjugate containing GDH specific antibody and polyclonal antibodies specific to toxins A and B. Diluent/conjugate mixture can be incubated for up to 24 hours. Moreover, components have to be added step wise, after waiting time - 15 minute incubation followed by 10 minute reaction period after which the result can be interpreted. Test kits have limited shelf life (expiration date) and need to be stored between 2-8°C, although they must be at room temperature at time of use. Tests cannot be reused. Faecal specimens for testing should ideally be no older than 24 hours, but can be tested up to 72 hours following collection. Frozen samples may lose activity due to the freeze-thaw cycle and degradation of toxins. Such frozen samples must also be thawed at room temperature. Such an assay kit is known from US 5,695,375. Using gas chromatography-mass spectrometry (GCMS), Probert et al. [A novel method for rapidly diagnosing the causes of diarrhoea. Gut 53, 58-61,2003] found characteristic patterns of volatile organic compounds (VOCs) related to several important causes of infectious diarrhoea including rotavirus, Campylobacter and C. difficile. In a later publication [Volatile organic compounds from feces and their potential for diagnosis of gastrointestinal disease. FASEB J 21, 1675-1688, 2007] the same team extended their work to the investigation of several hundred faecal VOCs and their potential for diagnosis of ulcerative colitis as well as Campylobacter jejuni and C. difficile. Compounds most strongly indicative of C. difficile were 2-furancarboxaldehyde (2FC) and 5-methyl-2- furancaboxaldehyde (5M2FC).
4-methyl-phenol (p-cresol) is also known to have an association with C. difficile. WO2004/008953 to Probert and Ratcliff mentions efforts 'made to rapidly identify C. difficile in cultures by presence of p-cresol and caproic acid', referring to Johnson LL, McFarland LV, Dealing P, Raisys V, Schoenknecht FD in Identification of Clostridium difficile in stool specimens by culture-enhanced gas-liquid chromatography, J Clin Microbiol 1989 ; 27: 2218- 21 and Nonhoff C, Struelens MJ, Serruys E. Evaluation of gas- liquid chromatography (GLC) for rapid detection of Clostridium difficle in faecal specimens. Acta Clinica Belgica 1995; 50: 76-80. However, these references only confirm a relationship between the presence of C. difficile and phenylacetic acid, isocaproic acid and hydrocinnamic acid. Moreover, p-cresol is known to be present in the faecal headspace of healthy beings - see e.g. V De Preter, G Van Staeyen, D Esser, P Rutgeerts and K Verbeke. Development of a screening method to determine the pattern of fermentation metabolites in faecal samples using on-line purge -and-trap gas chromatographic-mass spectrometric analysis. J. Chromatog. A 1216, 1476-1483, 2009. The present inventors have previously disclosed the development of a device for the diagnosis of C. Difficile Associated Diarrhoea employing the detection in faecal headspace of volatile organic compounds produced by C. Difficile and based on quantitative analysis to determine the concentrations of p-cresol produced in faecal samples from infected and non- infected patients.
DISCLOSURE OF INVENTION
According to a first aspect of the present invention, there is provided a method of analysing a faecal sample, the method comprising the steps of:
providing a sealed chamber having an inlet and an outlet;
placing a faecal sample in the chamber; thereafter
feeding a volume of gas through the inlet and into contact with the faecal sample; thereafter
exhausting said volume of gas through the outlet; and thereafter
analysing the chemical composition of said volume of gas.
The method may comprise the step of continuously feeding volumes of gas into the chamber and simultaneously continuously exhausting volumes of gas from the chamber, i.e. there may be a continuous flow of gas through the chamber. The continuous flow of gas may be at a rate of about 100ml per minute.
The method may comprise the step of analysing the continuously exhausted volumes of gas, i.e. continually analysing the flow of gas exhausted from the chamber.
The method may comprise the step of incubating the faecal sample prior to feeding gas into the chamber. The method may comprise the step of incubating the sample at about 40 ° C. The method may comprise the step of incubating the sample for about 5 minutes prior to feeding gas into the chamber.
The method may comprise the step of sucking gas out of the chamber.
The method may comprise the step of sucking ambient air in to the chamber. The ambient air may be filtered prior to entry into the chamber. The ambient air may be filtered to remove hydrocarbons.
The outlet may be fluidly connectable to an analyser. The outlet may also be fluidly connectable to an analyser bypass path. Analyser and analyser bypass path may both be fluidly connected to a suction pump. The method may comprise the step of switching the fluid connection of the outlet between the analyser and the bypass path without interrupting the continuous flow of gas through the chamber.
The method may comprise the step of generating a signal indicative of the presence of the Clostridium difficile bacterium in the faecal sample in dependence on the concentration indicated by the analysis of one or more compounds selected from the group of 1-Propanol, Butanal 3 -methyl, Propanoic acid ethyl ester, Isovaleric acid, 4-Heptanone, Phenol 4-methyl, Indole and Hexanoic acid, in particular Phenol 4-methyl (p-cresol).
The method may comprise the step of generating a signal indicative of the presence of the Clostridium difficile bacterium in the faecal sample where the concentration indicated by the analysis of the one or more compounds selected from the group of 1-Propanol, Butanal 3 -methyl, Propanoic acid ethyl ester, Isovaleric acid, 4-Heptanone, Phenol 4-methyl and Indole exceeds a predetermined threshold for that compound.
The method may comprise the step of generating a signal indicative of the presence of the Clostridium difficile bacterium in the faecal sample where the concentration of Hexanoic acid indicated by the analysis is lower than a predetermined threshold. According to a second aspect of the invention, there is provided a faecal sample analyser, comprising:
a faecal sample chamber having an inlet and an outlet;
a pump configured to feed a volume of gas through the inlet, into contact with a faecal sample and to exhaust said volume of gas through the outlet; and
an analyser for analysing the chemical composition of said volume of gas.
The apparatus aspects of the invention can be particularlised using features of the method described above.
BRIEF DESCRIPTION OF DRAWINGS
An embodiment of the invention will now be described by way of example with reference to the accompanying drawings, in which:
Figure 1 is a schematic illustrating an embodiment of the present invention;
Figures 2-8 and 9 are box and whisker plots for 'presence' and 'absence' compounds respectively.
Figure 10 is a flow chart illustrating the method according to the invention.
DETAILED DESCRIPTION OF SPECIFIC EMBODFMENTS
Referring to figure 1, the apparatus comprises a chamber 20 defined by a container 28 - in this case a 100ml Duran™ flask - and sealed except for a gas inlet 22 and a gas outlet 24 formed in a seal 26. Seal 26 can be temporarily broken to allow a cup 21 - typically of 2cm diameter - filled with a faecal sample S to be placed on the floor 30 of the chamber. Filling the cup 21 to the brim (and scraping the surface of the sample with a scraper ('doctor blade') as necessary to remove any excess above the brim) ensures a consistent, known sample area A.
Container 28 slots into an insulated heating jacket 36 comprising an aluminium mesh 38 surrounded by one or more heating elements 40 which are in turn surrounded by insulation 42. As indicated by dashed lines, controller 44 controls the electricity supply to the elements in dependence on the signal from a temperature sensor 46 located at the base of the jacket, adjacent the base of the container and the floor of the chamber. In the example shown, the temperature adjacent the base of the container is maintained at approximately 40°C.
Following incubation of the sample S at this temperature (typically for a short period of time of approximately 5 minutes), a pump 34 is started. As indicated by arrows, pump 34 sucks ambient air A through a hydrocarbon trap 32 and gas inlet 22 into the chamber 20 where it contacts the faecal sample S and is then exhausted through gas outlet 24. Trap 32 removes hydrocarbon-containing contaminants which would otherwise interfere with subsequent analysis. Pump 34 provides a continuous, constant flow of 100 ml min"1 at all times so as to allow the sample to reach equilibrium. Such an arrangement also allows data on the rate of emission from the sample, e.g. in milligrams per area A of sample per unit of time, to be collected as necessary.
The exhausted gas is drawn via valves Vi and V2 either through an analyser 48 or through a bypass tube 50 and thence to the pump 34 where it is exhausted to atmosphere as indicated at Z.
In the embodiment shown, analyser 48 is a sampling thermal desorption (TD) tube. Gas is allowed to flow through the tube for a period (in the example shown, five minutes) following which, by operation of valves Vi and V2, the flow is switched to a bypass tube 50 as indicated by arrows 52. Bypass tube may have the same flow resistance characteristics as the TD tube so as not to upset the flow through the chamber. Switching allows the sampling thermal desorption tube 48 to be removed for analysis. A fresh sampling tube can be inserted and/or the sample can be removed from the chamber and a fresh sample can be analysed and/or a new container can be used, as appropriate.
In the example shown, analysis of the TD tube is as follows. An internal standard solution comprising 50ng d8-toluene (Supelco Cat No. 48593) in methanol is added to each tube according to the manufacturer's instructions (Markes International Ltd, Llantrisant, UK). Samples are analysed by thermal desorption gas chromatography mass spectrometry (TD-GC-MS), the analytical instrumentation comprising a Series 2 Unity thermal desorber with Series 2 Ultra autosampler connected to an Agilent model 7890 gas chromatograph and ALMSCO Bench-ToF time-of-flight mass spectrometer (Markes International, Llantrisant, UK), the gas chromatograph being fitted with a DB5 column of dimensions 60m x 0.4mm x 0.25mm (Agilent Technologies UK Limited, Stockport, UK). The carrier gas is CP grade helium (BOC gases, Guildford, UK) passed through a combined trap for removal of hydrocarbons, oxygen and water vapour.
TD tubes are initially purged for 1 minute in order to remove air and water vapour then desorbed at 300° C for 8 minutes onto a secondary cold trap (Materials emission trap,
U-T12ME-2S) initially maintained at -10° C. Once desorption is complete, the secondary trap is heated at the maximum available rate to 300° C and maintained for 3 minutes whilst the effluent is transferred to the GC via a transfer line maintained at 150° C. Constant-flow operation is used, at 1.2 ml.min"1. Initial GC temperature is 35 ° C held for 1 minute, then increased at 2° C/min to 75 ° C, 5 ° C/min to 140° C and 10° C/min to 300° C where it is held for 10 minutes. The eluted products are transferred to the MS via a line maintained at 200° C where they are subjected to electron ionisation, the ion source being maintained at
200° C. The MS is operated from 20 - 450 amu at an effective scan rate of approximately 2.2 Hz. Compound identification is achieved using Chemstation (enhanced data analysis) software and the National Institute of Standards and Technology (NIST) mass spectral library. Quantification is achieved by comparing the area of each compound peak with the peak area associated with the known amount of d8-toluene.
Using the above techniques, it has been discovered by the present inventors that the invention may be used to identify the presence or absence of the Clostridium difficile bacterium in a faecal sample. Specifically, it has been discovered that there is a statistically significant correlation between the presence of Clostridium difficile bacterium in a faecal sample and the concentration above a certain level in the gas analysed according to the invention of one or more of the compounds 1-Propanol, Butanal 3 -methyl, Propanoic acid ethyl ester, Isovaleric acid, 4-Heptanone, Phenol 4-methyl and Indole. It has also been discovered that there is a statistically significant correlation between the presence of Clostridium difficile bacterium in a faecal sample and the concentration below a certain level in the gas analysed according to the invention of Hexanoic acid.
The correlations are illustrated in the box and whisker plots of figures 2-8 (for the first seven 'presence' compounds) and figure 9 (for the eighth 'absence' compound). The plots each show a difference, significant at the 5% level of probability, in median concentration of a compound between a group (I) of clinical samples known to be negative for C Diffi; and a group (II) of clinical samples known to be positive for C Diffi. There is also shown a group (III) of fresh controls (from healthy volunteers); and a group (IV) of frozen controls (from healthy volunteers). Of the seven 'presence' compounds, Phenol 4 methyl- is particularly advantageous as there is (a) a large difference of around 7.5000 in median concentration between group I and group II, and (b) no overlap between the boxes defining the upper and lower quartiles of group I and group II. Table 1 shows the parameters of the corresponding receiver operator characteristic (ROC), the standard error being under the nonparametric assumption, the null hypothesis for the asymptotic significance being true area = 0.5. As is known, the ROC characteristic indicates the overall performance of a compound in distinguishing between infected and non- infected samples, an ideal test having an area of 1 (or zero for an absence marker). It will be seen that the presence markers according to the present invention have a lower bound (95% confidence interval) greater than or equal to 0.6 while the absence marker according to the present invention has an upper bound less than or equal to 0.4. For avoidance of doubt, the Chemical Abstract Service (CAS) identification number for each compound is also indicated.
Based on these presence markers, the present invention can be used to identify the presence of the Clostridium difficile bacterium in a faecal sample as illustrated in figure 10. The sample is placed in the chamber, incubated and subject to gas flow as discussed above. The exhausted gas is then analysed to establish values of concentration of 1-Propanol, Butanal 3 -methyl, Propanoic acid ethyl ester, Isovaleric acid, 4-Heptanone, Phenol 4-methyl and Indole. These values are then compared with predetermined thresholds for each compound and, where a value exceeds a threshold, a signal is generated indicative of the presence of C.Diff in the sample. The exhausted gas is also analysed to establish a value of concentration of Hexanoic acid and, where a value is lower than a predetermined threshold, a signal is generated indicative of the presence of C.Diff in the sample.
It should be understood that this invention has been described by way of examples only and that a wide variety of modifications can be made without departing from the scope of the invention. TABLE 1
Figure imgf000011_0001

Claims

1. Method of analysing a faecal sample, the method comprising the steps of:
providing a sealed chamber having an inlet and an outlet;
placing a faecal sample in the chamber; thereafter
feeding a volume of gas through the inlet and into contact with the faecal sample; thereafter
exhausting said volume of gas through the outlet; and thereafter
analysing the chemical composition of said volume of gas.
2. Method according to claim 1 and comprising the step of continuously feeding volumes of gas into the chamber and simultaneously continuously exhausting volumes of gas from the chamber.
3. Method according to claim 2, wherein the continuous flow of gas through the chamber is at a rate of about 100ml per minute.
4. Method according to claim 2 or claim 3 and comprising the step of analysing the continuously exhausted volumes of gas.
5. Method according to any preceding claim and comprising the step of incubating the faecal sample prior to feeding gas into the chamber.
6. Method according to claim 5 and comprising the step of incubating the sample at about 40 ° C.
7. Method according to claim 6 and comprising the step of incubating the sample for about 5 minutes prior to feeding gas into the chamber.
8. Method according to any preceding claim and comprising the step of sucking gas out of the chamber.
9. Method according to any preceding claim and comprising the step of sucking ambient air in to the chamber.
10. Method according to claim 9, wherein the ambient air is filtered prior to entry into the chamber.
11. Method according to claim 10, wherein the ambient air is filtered to remove hydrocarbons.
12. Method according to any preceding claim, wherein the outlet is fluidly connectable to an analyser.
13. Method according to claim 12, wherein the outlet is also fluidly connectable to an analyser bypass path.
14. Method according to claim 13, wherein analyser and analyser bypass path are both fluidly connected to a suction pump.
15. Method according to claim 13 or claim 14, wherein the method comprises the step of 5 switching the fluid connection of the outlet between the analyser and the bypass path without interrupting the continuous flow of gas through the chamber.
16. Method according to any preceding claim and comprising the step of generating a signal indicative of the presence of the Clostridium difficile bacterium in the faecal sample in
10 dependence on the concentration indicated by the analysis of one or more compounds selected from the group of 1-Propanol, Butanal 3 -methyl, Propanoic acid ethyl ester, Isovaleric acid, 4-Heptanone, Phenol 4-methyl, Indole and Hexanoic acid, in particular Phenol 4-methyl (p-cresol).
15 17. Method according to claim 16 and comprising the step of generating a signal indicative of the presence of the Clostridium difficile bacterium in the faecal sample where the concentration indicated by the analysis of the one or more compounds selected from the group of 1-Propanol, Butanal 3 -methyl, Propanoic acid ethyl ester, Isovaleric acid, 4- Heptanone, Phenol 4-methyl and Indole exceeds a predetermined threshold for that 0 compound.
18. Method according to claim 16 and comprising the step of generating a signal indicative of the presence of the Clostridium difficile bacterium in the faecal sample where the concentration of Hexanoic acid indicated by the analysis is lower than a predetermined threshold.
19. Faecal sample analyser, comprising:
a faecal sample chamber having an inlet and an outlet;
a pump configured to feed a volume of gas through the inlet, into contact with a faecal sample and to exhaust said volume of gas through the outlet; and
an analyser for analysing the chemical composition of said volume of gas.
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