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WO2009037492A2 - Marqueurs - Google Patents

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Publication number
WO2009037492A2
WO2009037492A2 PCT/GB2008/003214 GB2008003214W WO2009037492A2 WO 2009037492 A2 WO2009037492 A2 WO 2009037492A2 GB 2008003214 W GB2008003214 W GB 2008003214W WO 2009037492 A2 WO2009037492 A2 WO 2009037492A2
Authority
WO
WIPO (PCT)
Prior art keywords
sample
compounds
mycobacteria
methyl
tuberculosis
Prior art date
Application number
PCT/GB2008/003214
Other languages
English (en)
Other versions
WO2009037492A3 (fr
Inventor
Ruth Mcnerney
Claire Turner
Original Assignee
London School Of Hygiene & Tropical Medicine
Cranfield University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by London School Of Hygiene & Tropical Medicine, Cranfield University filed Critical London School Of Hygiene & Tropical Medicine
Publication of WO2009037492A2 publication Critical patent/WO2009037492A2/fr
Publication of WO2009037492A3 publication Critical patent/WO2009037492A3/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor

Definitions

  • the invention relates to markers for use in the detection of the presence of Mycobacteria especially Mycobacterium tuberculosis, particularly markers in the volatile organic compounds emitted during the growth of the bacteria.
  • Mycobacterium tuberculosis which causes disease in humans and, less frequently in animals, and Mycobacterium bovis which is generally responsible for causing tuberculosis in animals, especially cattle and badgers.
  • VOCs volatile organic compounds
  • a method for confirming the presence of Mycobacteria in a sample comprising testing the sample or gases emitted by the sample for the presence of one or more of the following compounds: dimethyl sulfide; ethanethiol; 3-methyl-l-butanol; 2-methyl-l-propanol; pentane; 2-butanone; 2,3-dimethyl-butane; 2-methyl-l-butanol; pentanal; 2-methylbutanoic acid methyl ester; and phenyl ethyl alcohol; or a metabolite of any of these compounds; wherein the presence of an increased amount of one or more of the compounds or a metabolite thereof when compared with a sample that does not contain Mycobacteria is indicative of the presence of Mycobacteria.
  • Also provided is a method of diagnosing a subject as being infected with Mycobacteria comprising testing a sample obtained from the subject, or gases emitted by the sample, for the presence of one or more of the following compounds: dimethyl sulfide; ethanethiol; 3-methyl-l-butanol; 2-methyl-l-propanol; pentane; 2-butanone;
  • Mycobacteria include a complex of bacteria called the Mycobacterium tuberculosis complex, which includes Mycobacterium tuberculosis, M. bovis, M. africanum, Bacille Calmette- Guerin (BCG), M canettii, M. caprae, M. pinnipedii and M. microti. It is preferable that the species of Mycobacteria identified is from the Mycobacterium tuberculosis complex, and especially that it is M. tuberculosis or M. bovis.
  • Mycobacterium tuberculosis complex which includes Mycobacterium tuberculosis, M. bovis, M. africanum, Bacille Calmette- Guerin (BCG), M canettii, M. caprae, M. pinnipedii and M. microti. It is preferable that the species of Mycobacteria identified is from the Mycobacterium tuberculosis complex, and especially that it is M. tuberculosis or M
  • M. tuberculosis and M. bovis are the bacteria that cause most cases of tuberculosis in man and animals respectively.
  • the bacteria are well known in the art.
  • the sample may be any sample in which Mycobacteria could exist or that has been in close proximity to a source of Mycobacteria.
  • the sample could be a culture of M. tuberculosis or M. bovis or could be a sample obtained from a subject thought to be infected with Mycobacteria.
  • the sample is any sample from that subject that could contain Mycobacteria, such as but not limited to breath, blood, sputum, saliva, pleural, peritoneal, urine, faeces, bone marrow, tissue biopsies, especially from the lymph nodes and other deep tissues such as kidneys.
  • the actual sample obtained may not itself contain Mycobacteria, but may also have simply been in close proximity to Mycobacteria.
  • gases emitted by the sample is used to refer to any gaseous material released by or emitted by the sample, especially during bacterial growth.
  • the compounds mentioned above are all volatile organic compounds. Volatile organic compounds are organic compounds that have high enough vapour pressures under normal conditions to significantly vaporize and enter the atmosphere. The term is well known in the art.
  • the structures of the compounds mentioned above are provided in figure 1.
  • the compounds mentioned are metabolites produced by Mycobacteria, especially during growth. As mentioned above, further metabolites of the compounds mentioned may also be useful as markers of the presence of Mycobacteria.
  • metabolites of the compounds when broken down by Mycobacteria are most useful.
  • Such metabolites may include sulphur containing compounds.
  • Sulphur containing compounds may be detected by any appropriate means, a number of which are well known in the art and include x ray fluorescence, combustion ultraviolet fluorescence, combustion ion chromatography, oxidative pyrolysis and colorimetric detection, atomic absorbtion spectrometry, and atomic fluorescence.
  • the term "increased amount” preferably means that a statistically significantly increased amount of the compound is contained or released by the sample containing Mycobacteria than by a similar sample that does not contain Mycobacteria.
  • the two samples to be compared should be as similar as possible, except for the Mycobacteria, that is to say the same type of samples should be compared, e.g. breath with breath, blood with blood etc.
  • the sample containing Mycobacteria, or gas emitted by the sample contains at least 30% more of the compound in question, more preferably at least 40% more, more preferably at least 50% more, more preferably at least 60% more, more preferably at least 70% more and even more preferably at least 80% more.
  • the term "increased amount” may mean that a statistically significantly increased amount of the compound is contained or released by the sample when compared with other compounds contained or released by the sample.
  • the amount of a particular compound contained in or released by a particular sample may depend on the size of the sample, the way and length of time that sample has been stored and other variables. When considering the amount of a relevant compound, it may be more accurate to compare the level of that compound with the level of other compounds in or released by that sample. Accordingly, an increased proportion of a compound in relation to other compounds in the same sample may also be considered an increased amount.
  • the proportion of the relevant compound or compounds in or released by the sample is increased by at least 10%, 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50% and even more preferably at least 60%.
  • the term increased amount may mean that there is a significantly increased amount when compared with a number of other samples.
  • the method preferably comprises identifying the presence of at least two of the compounds or metabolites thereof, more preferably at least three, more preferably at least four, more preferably at least five, more preferably at least six, more preferably at least seven, more preferably at least eight, more preferably at least nine, more preferably at least ten, most preferably all the compounds or metabolites thereof.
  • the compounds may be identified in any combination. It is particularly preferably that at least phenylethyl alcohol is identified as being present in an increased amount.
  • the method may be carried out in any appropriate manner.
  • the compounds released by the sample may be collected by storing the sample in a fluid tight container and removing some of the gas released by the sample into the container. The gas may be removed once or repeatedly and the method repeated.
  • the container in which the sample is stored may contain a probe that may be used to detect the compounds.
  • the sample is breath, the sample itself maybe tested for the presence of the compounds, the breath containing compounds emitted by bacteria in the subject from whom the breath sample has been obtained. Alternatively, the sample itself may be tested, in which case, the compounds may be in solution.
  • the presence of the compounds may be assessed using standard methods, such as gas chromatography to separate the compounds, mass spectrometry, detection using a surface wave acoustic detector (SAW) or adsorbtion to sensors, such as in an electronic nose.
  • the first method of the invention can be used, for example, to confirm the presence of Mycobacteria in, for example, a culture sample. This is particularly useful as the method can be used to test the effectiveness of antibiotics against Mycobacteria. For example, a sample can be tested for the presence of Mycobacteria using the method of the invention, then an antibiotic may be applied to the sample and the sample tested again for the presence of Mycobacteria. If the Mycobacteria remains, the antibiotic is not effective against Mycobacteria.
  • the second method may be used to diagnose an infection with Mycobacterium tuberculosis in a subject, especially a subject exhibiting symptoms of tuberculosis or who has had a positive skin test for infection with Mycobacterium tuberculosis, using a sample obtained from the subject. It is useful to carry out such a method on a sample obtained from a subject who has had a positive skin test for Mycobacterium tuberculosis, because a skin test does not differentiate between latent and active tuberculosis.
  • the marker compounds used in the present method are emitted by the bacteria during growth. Hence, such compounds should only be emitted when the disease is active.
  • the methods may be used repeatedly to, for example, provide further confirmation of a diagnosis.
  • the method of diagnosis may be carried out on a number of samples or on both a sample and gases emitted by that sample.
  • the detection method used to identify the compounds may be varied, again to provide further confirmation of the result.
  • Figure 1 shows the structures of the markers identified.
  • Example 1 Mycobacterium bovis, Mycobacterium smegmatis and Mycobacterium tuberculosis were obtained and grown over a period of time in sealed bottles with headspace.
  • the amount of phenylethyl alcohol was observed to increase over time as cultures grew, but decreased after cultures stopped growing.
  • the inventor has demonstrated that volatile organic compounds emitted by growing bacteria can be used to determine susceptibility to chemical inhibitors/antimicrobials.
  • BCG was sub-cultured onto Lowenstein Jensen slopes containing 500 ⁇ g/ml PNB, and Lowenstein Jensen without addition of the inhibitor (Slopes supplied by Media for Mycobacteria, Glamorgan, Wales).
  • Mycobacterium smegmatis an environmental nonpathogenic bacteria that is not susceptible to PNB was also tested.
  • Method A set of three solid, sloped cultures of M. bovis BCG in glass universal tubes were placed in each of three volatile-free collapsible sample bags (in this case made of Nalophan) and connected to a sampling device. Any appropriate sampling device may be used, such as an automated thermal desorption (ATD) tube or selected ion flow tube mass spectrometry (SIFT-MS) instrument).
  • ATD automated thermal desorption
  • SIFT-MS selected ion flow tube mass spectrometry
  • the bags were sealed. Once inside the sample bags, the lids were removed from the universal tubes. Hydrocarbon free air was then added to each of the bags, which were then placed in an incubator at 37 0 C to allow the generation of headspace from the cultures and the control medium.
  • a culture bag and control bag was analysed directly using SIFT- MS at 37 0 C using all three precursor ions. 700 ml headspace air from each bag was then pumped across a thermal desorption tube, and subsequently analysed by gas chromatography- mass spectrometry thermal desorption (GC-MS-TD).
  • GC-MS-TD gas chromatography- mass spectrometry thermal desorption
  • ammonia is greatly reduced in the culture headspace so is being uptaken greatly by the cultures.
  • potential marker compounds for M. bovis BCG (present in M. bovis culture headspace, absent in medium headspace) are as follows: Dimethyl sulfide ethanethiol
  • Phenylethyl alcohol was also identified by z-nose.

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Toxicology (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Other Investigation Or Analysis Of Materials By Electrical Means (AREA)

Abstract

La présente invention concerne l'utilisation de composés organiques volatils en tant que marqueurs dans la détection de la présence de Mycobactéries, notamment Mycobacterium tuberculosis. L'invention propose un grand nombre de composés utiles comme marqueurs. Ces composés sont fréquemment émis par les échantillons contenant des Mycobactéries et leur présence, en particulier à des niveaux élevés, peut être utilisée pour détecter la présence des bactéries. L'invention concerne également un test diagnostique pour la tuberculose, fondé sur la présence des bactéries.
PCT/GB2008/003214 2007-09-21 2008-09-22 Marqueurs WO2009037492A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB0718496.3 2007-09-21
GBGB0718496.3A GB0718496D0 (en) 2007-09-21 2007-09-21 Markers

Publications (2)

Publication Number Publication Date
WO2009037492A2 true WO2009037492A2 (fr) 2009-03-26
WO2009037492A3 WO2009037492A3 (fr) 2009-06-11

Family

ID=38670334

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2008/003214 WO2009037492A2 (fr) 2007-09-21 2008-09-22 Marqueurs

Country Status (2)

Country Link
GB (1) GB0718496D0 (fr)
WO (1) WO2009037492A2 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010126856A1 (fr) * 2009-04-27 2010-11-04 The Charles Stark Draper Laboratory, Inc. Détection rapide de composés organiques volatils pour l'identification de mycobacterium tuberculosis dans un échantillon
US10058823B2 (en) 2013-06-03 2018-08-28 Basf Se Membranes

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
KHOGA JAMAL M ET AL: "Fly-attracting volatiles produced by Rhodococcus fascians and Mycobacterium aurum isolated from myiatic lesions of sheep" JOURNAL OF MICROBIOLOGICAL METHODS, vol. 48, no. 2-3, February 2002 (2002-02), pages 281-287, XP002510745 ISSN: 0167-7012 *
PHILLIPS ET AL: "Volatile biomarkers of pulmonary tuberculosis in the breath" TUBERCULOSIS, ELSEVIER, GB, vol. 87, no. 1, January 2007 (2007-01), pages 44-52, XP005751050 ISSN: 1472-9792 cited in the application *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010126856A1 (fr) * 2009-04-27 2010-11-04 The Charles Stark Draper Laboratory, Inc. Détection rapide de composés organiques volatils pour l'identification de mycobacterium tuberculosis dans un échantillon
US8518663B2 (en) 2009-04-27 2013-08-27 The Charles Stark Draper Laboratory, Inc. Rapid detection of volatile organic compounds for identification of Mycobacterium tuberculosis in a sample
US10058823B2 (en) 2013-06-03 2018-08-28 Basf Se Membranes

Also Published As

Publication number Publication date
GB0718496D0 (en) 2007-10-31
WO2009037492A3 (fr) 2009-06-11

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