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EP2205970A1 - Détection de composés volatils en tant que marqueurs de la tuberculose due aux mycobactéries - Google Patents

Détection de composés volatils en tant que marqueurs de la tuberculose due aux mycobactéries

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Publication number
EP2205970A1
EP2205970A1 EP08835643A EP08835643A EP2205970A1 EP 2205970 A1 EP2205970 A1 EP 2205970A1 EP 08835643 A EP08835643 A EP 08835643A EP 08835643 A EP08835643 A EP 08835643A EP 2205970 A1 EP2205970 A1 EP 2205970A1
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EP
European Patent Office
Prior art keywords
mycobacterium
sample
acid
methyl
microorganism
Prior art date
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EP08835643A
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German (de)
English (en)
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EP2205970A4 (fr
Inventor
Mona Syhre
Stephen T. Chambers
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University of Otago
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University of Otago
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Publication of EP2205970A1 publication Critical patent/EP2205970A1/fr
Publication of EP2205970A4 publication Critical patent/EP2205970A4/fr
Withdrawn legal-status Critical Current

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    • 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
    • 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
    • G01N2333/35—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Mycobacteriaceae (F)

Definitions

  • the invention relates to the use of biomarkers for identifying bacterial pathogens.
  • the invention relates to the detection and identification of certain volatile compounds as markers for Mycobacterium tuberculosis and/or Mycobacterium bovis in an individual or from a culture by analysis of a gaseous sample.
  • microorganisms produce by-products as a result of their normal metabolism.
  • the ability of different organisms to metabolise different substrates in order to satisfy their energy and nutritional requirements is fundamental to laboratory microbiology, and forms the basis of many rapid identification tests.
  • the metabolites produced by a single species can vary widely, depending upon the growth substrate, conditions (temperature, oxygen availability), and the age of the culture itself.
  • MVOCs Microbial volatile organic compounds
  • Some MVOCs have important health and economic implications in these fields.
  • MVOCs have been associated with spoilage in stored crops and foodstuffs, where they may be responsible for tainting, "off flavours, discolouring of products, or toxicity.
  • Profiles of MVOCs are increasingly being found to be unique to the species or strain. This should not seem surprising, as it is the different biochemical ability of different microbes that gives them most of their identifiable and distinguishing traits.
  • pulmonary tuberculosis has added greatly to the burden of poor health in developing countries. TB now kills one person every 18 seconds worldwide. TB is a difficult disease to diagnose, due mainly to the difficulty in culturing this slow- growing organism in the laboratory. A complete medical evaluation for TB must include a medical history, a chest X-ray, and a physical examination. Diagnosis of TB still relies on sputum staining for acid-fast bacilli. This method is time consuming. It has high specificity, but poor sensitivity.
  • Fluorochrome staining with auramine-rhodamine is the preferred staining method because it is faster than the traditional methods in which Ziehl-Neelsen or Kinyoun (basic fuchsin dye) stains are used.
  • Smear examination is an easy and quick procedure - results should be available within 24 hours of specimen collection. However, smear examination permits only the presumptive diagnosis of TB because the AFB in a smear may be mycobacteria other than M. tuberculosis. Furthermore, many TB positive patients have negative AFB smears.
  • TB may also be diagnosed on the basis of clinical signs and symptoms in the absence of a positive culture. Culture examinations should be done on all specimens, regardless of AFB smear results.
  • the BACTEC Radiometric System or other recently developed liquid medium systems, enables detection of mycobacterial growth in 4 to 14 days.
  • nucleic acid probes can identify the species in 2 to 4 hours.
  • Nucleic acid probes specific for the M. tuberculosis complex, for M. avium, and for M. intracellular provide a rapid method of species identification.
  • High-performance liquid chromatography (HPLC) which detects differences in the spectrum of mycolic acids in the cell wall, is equally rapid and can identify most pathogenic mycobacterial species.
  • a test for inhibition by r-nitro-a- acetylamino-b-hydroxypropiophenone (NAP or NAP test) can identify M. tuberculosis in 3 to 4 days. If a solid medium and conventional biochemical tests are used, the isolation and identification of the organism can take 6 to 12 weeks.
  • NAA tests such as PCR and other methods for amplifying DNA and RNA, may facilitate rapid detection of microorganisms.
  • Commercial NAA kits for the identification of M. tuberculosis complex have been approved by the Food and Drug Administration (FDA) for use on processed clinical specimens. These tests perform worst where needed most. Specificity is inadequate when applied to smear- negative specimens and sensitivity is inadequate when applied to smear-positive specimens.
  • FDA Food and Drug Administration
  • the test is approved for use in conjunction with cultures for respiratory specimens that are positive for AFB on microscopy and were obtained from untreated patients. When used as approved, a positive NAA test result indicates a high likelihood of TB, but a negative result does not exclude TB.
  • MTD Mycobacterium Tuberculosis Direct
  • the BACTEC radiometric method which uses a liquid medium, is faster than conventional methods for determining susceptibility to first-line TB medications. Usually, susceptibility results can be obtained within 7-14 days of BACTEC inoculation. Conventional methods, which use solid media for growth, can take as long as 21 days after inoculation.
  • US 3,616,258 describes a diagnostic product for detection of niacin produced by M. tuberculosis, which product uses an impregnated paper strip.
  • WO 2006/079846 describes a method of detecting and identifying bacteria comprising the steps of collecting volatile bacterial products, subjecting the volatile products to a gas chromatography system employing a surface acoustic wave detector, and establishing chromatographic profiles for different bacteria.
  • the system is said to be suitable for use in the detection of TB and indicates that mycolic acids could be a potential marker.
  • Example 1 relates to the identification of TB, but this is a prophetic example teaching how this might performed. No specific markers for TB are described.
  • WO 2003/075745 describes a method for detecting a physiological condition in an animal by measuring a clinical diagnostic marker using an array of sensors to generate a sensor array response profile.
  • the detection and monitoring of TB is described as one possible medical application. However, there is no indication of how TB could be detected, or what markers could be used.
  • WO 2003/064994 describes a method of collecting and detecting compounds in a human breath sample using a hand held sample collector to absorb at least one breath compound. The collector can then be connected to a breath analyser. The method is described as an easy-to-use diagnostic method for the detection of TB. Further, it is stated that the composition and concentration of volatile compounds emitted from TB- infected cells in the lung is largely unknown, but the detection of tuberculostearic acid (TSA) in TB cultures and in sputum and serum samples of TB patients suggests the presence of characteristic metabolites that might be useful in diagnosing TB.
  • TSA tuberculostearic acid
  • WO 2005/079669 describes a method and apparatus for diagnosing various medical conditions by analysing breath.
  • the system uses phase measurement of absorption of IR radiation to allow very small concentrations of the diagnostic species to be measured.
  • An example is given of using the system to test ethane in a number of patients. It is merely speculated that this measurement could be a potential diagnostic for TB.
  • WO 2004/090534 describes a system for breath analysis using asymmetric ion mobility spectrometry.
  • TB is disclosed as a potential condition that could be identified using the system. However, no specific markers for TB are described.
  • the applicant has now identified a set of compounds which are unique identifiers for TB.
  • the same compounds have not been detected in vitro from other fungi and bacteria related to pulmonary diseases. These compounds represent a useful tool for the identification of Mycobacterium tuberculosis and Mycobacterium bovis from culture samples and also for breath diagnosis of TB.
  • a method for detecting a Mycobacterium microorganism by analysing a gas mixture for any one or more of methyl phenylacetate, methyl p-anisate, methyl nicotinate, and o-phenylanisole.
  • the invention provides a method of detecting a Mycobacterium microorganism in a patient comprising: a) obtaining a sample of breath of the patient; b) analysing the sample for the presence of any one or more of methyl phenylacetate, methyl p-anisate, methyl nicotinate, and o-phenylanisole; and c) determining whether a Mycobacterium microorganism is present in the patient.
  • the invention provides a method of detecting a Mycobacterium microorganism in an in vitro culture or a biological sample comprising: a) obtaining a sample from the headspace gas of the in vitro culture or biological sample; b) analysing the sample for the presence of any one or more of methyl phenylacetate, methyl p-anisate, methyl nicotinate, and o-phenylanisole; and c) determining whether a Mycobacterium microorganism is present in the in vitro culture or biological sample.
  • the invention provides a method for detecting a Mycobacterium microorganism by analysing a gas mixture for any one or more of phenyl acetic acid, anisic acid (preferably p-anisic acid), nicotinic acid and salicylic acid.
  • the invention provides a method of detecting a Mycobacterium microorganism in a patient comprising: a) obtaining a sample of breath of the patient; b) analysing the sample for the presence of any one or more of phenyl acetic acid, anisic acid (preferably p-anisic acid), nicotinic acid and salicylic acid; and c) determining whether a Mycobacterium microorganism is present in the patient.
  • the invention provides a method for detecting a Mycobacterium microorganism in an in vitro culture or a biological sample comprising: a) obtaining a sample from the headspace gas of the in vitro culture or biological sample; b) analysing the sample for the presence of any one or more of phenyl acetic acid, anisic acid (preferably p-anisic acid), nicotinic acid and salicylic acid; and c) determining whether a Mycobacterium microorganism is present in the in vitro culture or biological sample.
  • the invention provides a method for detecting a Mycobacterium microorganism by analysing a gas mixture for a derivative of any one or more of phenyl acetic acid, anisic acid (preferably p-anisic acid), nicotinic acid and salicylic acid.
  • the invention provides a method of detecting a Mycobacterium microorganism in a patient comprising: a) obtaining a sample of breath of the patient; b) analysing the sample for the presence of a derivative of any one or more of phenyl acetic acid, anisic acid (preferably p-anisic acid), nicotinic acid and salicylic acid; and c) determining whether a Mycobacterium microorganism is present in the patient.
  • the invention provides a method for detecting a Mycobacterium microorganism in an in vitro culture or a biological sample comprising:
  • the derivative is an ester, e.g. an alkyl, alkenyl or alkynyl ester, preferably an alkyl ester, e.g. a lower alkyl ester, e.g. a methyl ester.
  • the derivative of phenyl acetic acid, anisic acid (preferably p-anisic acid), nicotinic acid or salicylic acid may be a silyl derivative, e.g. a trimethylsilyl derivative.
  • a method for detecting a Mycobacterium microorganism by analysing a gas mixture for any one or more of methyl phenylacetate, . methyl p-anisate, methyl nicotinate, o-phenylanisole, phenyl acetic acid, anisic acid (preferably p-anisic acid), nicotinic acid and salicylic acid.
  • the invention provides a method of detecting a Mycobacterium microorganism in a patient comprising: a) obtaining a sample of breath of the patient; b) analysing the sample for the presence of any one or more of methyl phenylacetate, methyl p-anisate, methyl nicotinate, o-phenylanisole, phenyl acetic acid, anisic acid (preferably p-anisic acid), nicotinic acid and salicylic acid; and c) determining whether a Mycobacterium microorganism is present in the patient.
  • the invention provides a method for detecting a Mycobacterium microorganism in an in vitro culture or a biological sample comprising: a) obtaining a sample from the headspace gas of the in vitro culture or biological sample; b) analysing the sample for the presence of any one or more of methyl phenylacetate, methyl p-anisate, methyl nicotinate, o-phenylanisole, phenyl acetic acid, anisic acid (preferably p-anisic acid), nicotinic acid and salicylic acid; and c) determining whether a Mycobacterium microorganism is present in the in vitro culture or biological sample.
  • the method includes the following step (i), which may optionally be carried out after step a):
  • step b) is analysing the sample for the presence of the derivative of phenyl acetic acid, anisic acid (preferably p-anisic acid), nicotinic acid or salicylic acid.
  • the derivative is an ester, e.g. an alkyl, alkenyl or alkynyl ester, preferably an alkyl ester, e.g. a lower alkyl ester, e.g. a methyl ester.
  • the derivative of phenyl acetic acid, anisic acid (preferably p-anisic acid), nicotinic acid or salicylic acid may be a silyl derivative, e.g. a trimethylsilyl derivative.
  • the gas mixture is preferably analysed for both methyl p-anisate and methyl nicotinate. In some embodiments of the invention, it may be preferable to analyse the gas mixture for all of methyl phenylacetate, methyl p-anisate, methyl nicotinate, and o-phenylanisole. In other embodiments of the invention, it may be preferable to analyse the gas mixture for all of methyl phenylacetate, methyl p-anisate, methyl nicotinate, o-phenylanisole, phenyl acetic acid, anisic acid (preferably p-anisic acid), nicotinic acid and salicylic acid.
  • the in vitro culture or biological sample may be a liquid or a solid sample. Typically it is a liquid sample, e.g. sputum, blood, serum or lung fluid.
  • the biological sample is preferably sputum, but may also be lung fluid, serum or blood, or any other sample capable of analysis according to the invention.
  • gas mixture is analysed using GC-MS 1 e.g. as described in the Examples.
  • the invention provides an apparatus for detecting the presence of a Mycobacterium microorganism having: a) a gas sample inlet enabling introduction of a gas sample into the apparatus; b) a means for analysing the gas sample for the presence of any one or more of methyl phenylacetate, methyl p-anisate, methyl nicotinate, and o-phenylanisole; and c) a means for displaying information enabling the diagnosis of Mycobacterium tuberculosis, Mycobacterium africanum or Mycobacterium bovis, or a means for transmitting information to a device for displaying information enabling the diagnosis of Mycobacterium tuberculosis, Mycobacterium africanum or Mycobacterium bovis.
  • the invention provides an apparatus for detecting the presence of a Mycobacterium microorganism having: a) a gas sample inlet enabling introduction of a gas sample into the apparatus; b) a means for analysing the gas sample for the presence of any one or more of methyl phenylacetate, methyl p-anisate, methyl nicotinate, and o-phenylanisole; and c) a means for displaying information enabling the diagnosis of Mycobacterium tuberculosis or Mycobacterium bovis, or a means for transmitting information to a device for displaying information enabling the diagnosis of Mycobacterium tuberculosis or Mycobacterium bovis.
  • the invention provides a method of diagnosis of a mycobacterial infection in a patient, including detecting a Mycobacterium microorganism by any of the methods described above.
  • the gas sample is the headspace gas of a Mycobacterium culture, a sputum sample from a patient, e.g. a patient suspected of being infected with a mycobacterial infection, a blood sample from a patient, e.g. a patient suspected of being infected with a mycobacterial infection, a serum sample from a patient, e.g. a patient suspected of being infected with a mycobacterial infection or a lung fluid sample from a patient, e.g. a patient suspected of being infected with a mycobacterial infection.
  • the gas sample is a breath sample from a patient, e.g. a patient suspected of being infected with a mycobacterial infection.
  • the Mycobacterium microorganism is Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium smegmatis, Mycobacterium tuberculosis BCG, Mycobacterium avium, Mycobacterium leprae, Mycobacterium africanum or Mycobacterium ulcerans, especially Mycobacterium tuberculosis, Mycobacterium africanum or Mycobacterium bovis, in particular Mycobacterium tuberculosis.
  • Figure 1 shows growth dependent concentration curves of methyl p-anisate (TB5) on L ⁇ wenstein-Jensen medium.
  • Figure 2 shows the detection of methyl nicotinate (after derivatisation) in the headspace of serum samples of Mycobacterium tuberculosis-positive (p) and Mycobacterium tuberculosis-negative (n) patients.
  • the invention provides a method for detecting a Mycobacterium microorganism by analysing a gas mixture for any one or more of methyl phenylacetate, methyl p-anisate, methyl nicotinate, and o-phenylanisole.
  • the invention also provides a method for detecting a Mycobacterium microorganism by analysing a gas mixture for any one or more of nicotinic acid, anisic acid (preferably p- anisic acid), phenyl acetic acid and salicylic acid.
  • the invention also provides a method for detecting a Mycobacterium microorganism by analysing a gas mixture for any one or more of nicotinic acid, anisic acid (preferably p- anisic acid), phenyl acetic acid, salicylic acid, methyl phenylacetate, methyl p-anisate, methyl nicotinate, and o-phenylanisole.
  • anisic acid preferably p- anisic acid
  • phenyl acetic acid preferably p- anisic acid
  • salicylic acid methyl phenylacetate
  • methyl p-anisate methyl nicotinate
  • o-phenylanisole o-phenylanisole
  • the four volatile organic compounds methyl phenylacetate, methyl p-anisate, methyl nicotinate, and o-phenylanisole are unique biomarkers for Mycobacteria, e.g. TB, specifically Mycobacterium complex which comprises Mycobacterium tuberculosis and/or Mycobacterium bovis, particularly when detected in gas samples, e.g. a sample of breath of a patient or a sample of the headspace gas of an in vitro culture or biological sample.
  • ⁇ methyl phenylacetate (CAS RN 101-41-7) is also known as methyl alpha-toluate and phenyl acetic acid methyl ester.
  • ⁇ methyl p-anisate (CAS RN 121-98-2) is also known as methyl para-anisate, methyl anisate and methyl 4-methoxybenzoate.
  • ⁇ methyl nicotinate (CAS RN 93-60-7) is also known as methyl 3- pyridinecarboxylate.
  • ⁇ o-phenylanisole (CAS RN 86-26-0) is also known as 2-methoxybiphenyl.
  • ⁇ -anisic acid CAS RN 100-09-4
  • phenyl acetic acid CAS RN 100-79-2
  • salicylic acid CAS RN 69-72-7
  • biomarkers for Mycobacteria e.g. TB, specifically Mycobacterium complex which comprises Mycobacterium tuberculosis and/or Mycobacterium bovis, particularly when detected in gas samples, e.g. a sample of breath of a patient or a sample of the headspace gas of an in vitro culture or biological sample.
  • these acids may also be known by other names, corresponding to the other names shown above for their methyl esters.
  • SPME Solid Phase Micro Extraction
  • GC/MS Gas Chromatography/Mass Spectroscopy
  • the four compounds methyl phenylacetate, methyl p-anisate, methyl nicotinate, and o-phenylanisole were identified as a set of compounds which are unique for Mycobacterium. These compounds have not been detected in vitro from other fungi and bacteria related to pulmonary diseases.
  • the compounds are useful for the identification of Mycobacteria, e.g. Mycobacterium complex which comprises Mycobacterium tuberculosis and Mycobacterium bovis from culture, and also as useful specific markers for breath diagnosis of TB.
  • nicotinic acid p-anisic acid and phenyl acetic acid
  • salicylic acid detectable, either as the free acids or after in situ derivatisation, in patient samples, e.g. serum and sputum samples of patients suspected of being infected with a mycobacterial infection.
  • patient samples e.g. serum and sputum samples of patients suspected of being infected with a mycobacterial infection.
  • These acids are detectable, either as the free acids or after in situ derivatisation, in the headspace gas of samples of bodily fluid.
  • nicotinic acid p-anisic acid
  • phenyl acetic acid phenyl acetic acid
  • salicylic acid markers that are detectable in the headspace gas of samples of bodily fluid, e.g. sputum, lung fluid, serum, blood, of patients suspected of being infected with a mycobacterial infection, e.g. Mycobacterium tuberculosis or Mycobacterium bovis.
  • the acids can be converted to a variety of derivatives for detection as biomarkers.
  • a sample may be taken from an in vitro culture of a Mycobacterium microorganism, e.g. as described in the Examples section, or from a biological sample such as a sputum sample from a patient, e.g. a patient suspected of being infected with a mycobacterial infection; a blood sample from a patient, e.g. a patient suspected of being infected with a mycobacterial infection; a serum sample from a patient, e.g. a patient suspected of being infected with a mycobacterial infection; or a lung fluid sample from a patient, e.g. a patient suspected of being infected with a mycobacterial infection.
  • a biological sample such as a sputum sample from a patient, e.g. a patient suspected of being infected with a mycobacterial infection; a blood sample from a patient, e.g. a patient suspected of being infected with a mycobacterial infection; a serum sample from a patient,
  • the sample e.g. the sample taken from an in vitro culture of a Mycobacterium microorganism or the biological sample, may be prepared for sampling by placing in a suitable sample vial for collection of a sample of the headspace gas.
  • a sample of breath may be taken from a patient, e.g. a patient suspected of being infected with a mycobacterial infection.
  • the breath or the headspace gas from the culture or biological sample can be sampled, for example with a conditioned Solid Phase Micro Extraction (SPME) fibre or via another absorbent trap, such as Tenax.
  • SPME Solid Phase Micro Extraction
  • the sampled potion whether it be the breath sample or the headspace gas sample, is contacted with a suitable derivatising agent, e.g.
  • an alkylating agent e.g. trimethyl sulfonium hydroxide (TMSH) or trimethyl anilinium hydroxide (TMAH) or a silylating agent, e.g. N,O-bis(trimethylsilyl)acetamide (BSA) or N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA).
  • TMSH trimethyl sulfonium hydroxide
  • TMAH trimethyl anilinium hydroxide
  • silylating agent e.g. N,O-bis(trimethylsilyl)acetamide (BSA) or N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA).
  • the sample may be contacted with the derivatising agent while on the SPME fibre or on the absorbent trap.
  • the fibre is exposed directly into the spectrometer, e.g. into the hot injector of a gas chromatograph, e.g. as described in the Examples section.
  • This in situ derivatisation can give ester derivatives, e.g. alkyl ester derivatives such as methyl ester derivatives.
  • the acids can be converted as described above and in the Examples section, using a suitable alkylating agent such as TMSH or TMAH, to their corresponding alkyl esters, e.g. methyl esters.
  • TMSH TMSH
  • TMAH TMAH
  • Other alkylating agents can be used, such as, for example, diazomethane or methanolic BF 3 .
  • breath samples For analysis of patient breath samples, these samples can be delivered to the inlet of the spectrometer instrument. Alternatively, a breath sample can be collected, e.g. in a sample bag or other suitable container, for later delivery to the spectrometer instrument. It will also be appreciated by those skilled in the art that a number of different techniques are suitable for analysing the gas samples in the methods of the invention. One preferred technique is GC-MS, as described in the Examples.
  • PTR-MS can be used to analyse headspace gas samples of cell cultures of Pseudomonas aeruginosa and Steptococcus milleri.
  • the same technique can also be used to analyse patient breath samples for drug monitoring, as can SIFT-MS (A. Critchley, T. S. Elliott, G. Harrivson, C. A. Mayhew, J. M. Thompson and T. Worthington, International Journal of Mass Spectrometry, 239, 2004, 235-241).
  • SIFT-MS A. Critchley, T. S. Elliott, G. Harrivson, C. A. Mayhew, J. M. Thompson and T. Worthington, International Journal of Mass Spectrometry, 239, 2004, 235-241).
  • the technique may therefore be used to analyse headspace samples for the presence of volatile compounds, and thus finds application in the present methods for detecting the above-described biomarkers.
  • SIFT-MS can also be used to detect small molecules in patient breath samples (S. M. Abbott, J. B. Elder, P. Spanel, D. Smith, International Journal of Mass Spectrometry, 228, 2003, 655-665).
  • the specific volatile biomarkers enable the speedy diagnosis of M. tuberculosis and M. bovis relative to time consuming existing diagnostic methods. They provide increased sensitivity and specificity for these organisms.
  • the biomarkers enable the identification of M. tuberculosis, M.africanum and M.
  • bovis not only from culture samples, but also from sputum, lung fluid, serum and even blood and other body fluids.
  • the detection of the biomarkers by analysis of the headspace over these materials makes this method ideally suited for breath diagnosis of TB.
  • instrumentation capable of testing for trace amounts of volatile compounds in a gaseous sample such as those described above, e.g. electronic noses
  • diagnosis of TB by testing for the biomarkers on the breath of patients in both clinical and field environments is made possible by this invention.
  • patient includes both human and animal patients.
  • alkyl as used herein is intended to include both straight- and branched- chain alkyl groups.
  • lower alkyl means C 1 -C 6 alkyl.
  • alkyl groups include methyl group, ethyl group, n-propyl group, i-propyl group, n-butyl group, sec- butyl group i-butyl group, t-butyl group, n-pentyl group, 1 ,1-dimethylpropyl group, 1 ,2- dimethylpropyl group, 2,2-dimethylpropyl group, 1-ethylpropyl group, 2-ethylpropyl group, ⁇ -hexyl group and 1-methyl-2-ethyl propyl group.
  • Any alkyl group may be substituted or unsubstituted. Preferably it is unsubstituted. More preferably any alkyl group is an unsubstituted lower alkyl group.
  • alkenyl as used herein is intended to include both straight- and branched- chain alkenyl groups.
  • lower alkenyl means C 2 -C 6 alkenyl.
  • alkenyl groups include straight- or branched-chain enthenyl, propenyl, butenyl, pentenyl and hexenyl groups.
  • Any alkenyl group may be substituted or unsubstituted. Preferably it is unsubstituted. More preferably any alkenyl group is an unsubstituted lower alkenyl group.
  • alkynyl as used herein is intended to include both straight- and branched- chain alkynyl groups.
  • lower alkynyl means C 2 -C 6 alkynyl.
  • alkynyl groups include straight- or branched-chain ethynyl, propynyl, butynyl, pentynyl and hexynyl groups.
  • Any alkynyl group may be substituted or unsubstituted. Preferably it is unsubstituted. More preferably any alkynyl group is an unsubstituted lower alkynyl group.
  • headspace gas as used herein is intended to include the gas phase contained in a sample container, e.g. a chromatography sample vial, which also contains a liquid or solid sample.
  • Organisms were grown on Lowenstein Jensen/Glycerol, sheep blood agar and BacT/Alert®MP within sterile glass vials stoppered with airtight aluminium caps incorporating a teflon-coated rubber septum.
  • MB/BacT ® (0.5 ml_) reconstitution fluid (Biomerieux, Inc., Marcy I'Etoile, France) was added to each vial.
  • Freeze dried cultures were revived as recommended by the New Zealand Reference Culture Collection, Medical Section, and transferred onto the medium. Each medium was then incubated at 37 0 C for up to four weeks.
  • a suspension was made to 0.5 McFariand standard.
  • a 1 :10 6 dilution (equivalent to approximately 10 3 organisms) of this suspension (500 ⁇ l_) was introduced into the sealed culture vial by injecting through the septum onto the culture medium. The medium was then incubated at 37 0 C for up to four weeks or until there was visibly identifiable growth. Headspace probing was carried out at weekly intervals.
  • Verification of fully grown cultures was provided independently by the Microbiology Unit of the Canterbury Health Laboratories, Wales, New Zealand, using Fuchsin acid- fast stains (Ziehl-Neelsen) and microscopy.
  • Fuchsin acid- fast stains Ziehl-Neelsen
  • microscopy For time dependence of metabolite production, six dilutions (1:10 to 1:10 6 ) of a 0.5 McFariand standard of the reference strain of Mycobacterium tuberculosis were prepared. Each dilution (500 ⁇ l_) was injected through the septum onto all three media. The vials were incubated at 37 0 C and sampling was carried out every three days for four weeks.
  • a conditioned SPME fibre was exposed into culture vials for 12 hours and then desorbed directly in the injection port for 15 min.
  • the temperatures of the injector, ion trap, manifold and transfer line were 250, 200, 60 and 250 0 C, respectively.
  • a ZB-624 column was used. The oven program commenced at 60 0 C for 2 min, was raised to
  • Helium flow was set at a constant rate of 1.2 mUmin.
  • the split vent was opened to a ratio of 1:50 after 1 min. Fragmentation was performed in the El-mode as full scan which gave additional certainty. Further MS/MS fragmentation was used to increase sensitivity.
  • SPME fibres (colour code grey from Supelco, Bellefonte) were conditioned and then exposed via septa into the headspace of a 2 mL vial containing serum (0.3 ml_) or sputum (0.5 mL) and sampled for up to 10 hours.
  • a derivatisation step converted the free acid biomarkers present in the serum and sputum samples into their corresponding methyl esters.
  • Derivatisation was achieved in situ by exposing the fibre after the sampling step into the headspace of 0.2 M trimethyl sulfonium hydroxide (TMSH) for one minute.
  • TMSH trimethyl sulfonium hydroxide
  • the fibre was then retracted and again exposed into the hot injector of the gas chromatograph at 25O 0 C and analysed by GC- MS as described above and in M. Syhre and S. T. Chambers, Tuberculosis 88, 317 (2008).
  • the derivatisation took place in situ and the resulting methyl esters methyl phenylacetate, methyl p-anisate and methyl nicotinate were detected.
  • Methyl phenylacetate and methyl p-anisate were detected in the headspace of sputum samples after derivatisation. Methyl p-anisate and methyl nicotinate were detected in the headspace of serum samples.
  • Figure 2 shows the detection of methyl nicotinate (after derivatisation) in the headspace of serum samples of Mycobacterium tuberculosis-positive and Mycobacterium tuberculosis-negative patients.
  • the invention relates to the use of biomarkers for identifying bacterial pathogens.
  • the invention provides a method for detecting a Mycobacterium microorganism.

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Abstract

La présente invention concerne l'utilisation de biomarqueurs permettant l'identification d'agents pathogènes bactériens. En particulier, l'invention concerne la détection et l'identification de certains composés en tant que marqueurs pour des infections mycobactériennes chez un patient ou provenant d'une culture par l'analyse d'un échantillon gazeux.
EP08835643A 2007-10-05 2008-10-06 Détection de composés volatils en tant que marqueurs de la tuberculose due aux mycobactéries Withdrawn EP2205970A4 (fr)

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NZ562236A NZ562236A (en) 2007-10-05 2007-10-05 Detection of volatile compounds as markers for Mycobacteria tuberculosis
PCT/NZ2008/000261 WO2009045116A1 (fr) 2007-10-05 2008-10-06 Détection de composés volatils en tant que marqueurs de la tuberculose due aux mycobactéries

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WO2012143901A1 (fr) 2011-04-21 2012-10-26 North-West University Procédé pour permettre la distinction entre différents pathogènes
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WO2014070780A1 (fr) 2012-10-29 2014-05-08 University Of Utah Research Foundation Détecteurs à nanotubes fonctionnalisés et procédés associés
JP6529438B2 (ja) 2012-11-29 2019-06-12 インスメッド インコーポレイテッド 安定化されたバンコマイシン処方物
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WO2009045116A1 (fr) 2009-04-09
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EP2205970A4 (fr) 2011-07-06
NZ562236A (en) 2010-04-30

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