WO2001066790A2 - Procede rapide et sensible permettant de detecter un aspergillus fumigatus - Google Patents

Procede rapide et sensible permettant de detecter un aspergillus fumigatus Download PDF

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WO2001066790A2
WO2001066790A2 PCT/EP2001/002727 EP0102727W WO0166790A2 WO 2001066790 A2 WO2001066790 A2 WO 2001066790A2 EP 0102727 W EP0102727 W EP 0102727W WO 0166790 A2 WO0166790 A2 WO 0166790A2
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arabinopyranoside
membrane filter
substrate
membrane
sample
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PCT/EP2001/002727
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WO2001066790A3 (fr
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Hans J. Nelis
Tiene G. M. Bauters
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Universiteit Gent
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Priority to AU2001240692A priority Critical patent/AU2001240692A1/en
Publication of WO2001066790A2 publication Critical patent/WO2001066790A2/fr
Publication of WO2001066790A3 publication Critical patent/WO2001066790A3/fr

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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
    • 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
    • C12Q1/045—Culture media therefor
    • 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
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • 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
    • C12Q2334/00—O-linked chromogens for determinations of hydrolase enzymes, e.g. glycosidases, phosphatases, esterases
    • C12Q2334/20—Coumarin derivatives
    • C12Q2334/22—4-Methylumbelliferyl, i.e. beta-methylumbelliferone, 4MU
    • 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
    • G01N2333/37—Assays involving biological materials from specific organisms or of a specific nature from fungi
    • G01N2333/38—Assays involving biological materials from specific organisms or of a specific nature from fungi from Aspergillus
    • 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
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/924—Hydrolases (3) acting on glycosyl compounds (3.2)

Definitions

  • the present invention relates to the field of the specific and selective detection and identification of micro-organisms based on a specific enzymatic activity present in the microorganism to be detected. More particularly, the arabinopyranoside substrate cleaving activity present in specific micro-organisms is envisaged.
  • the present invention relates to methods for detecting micro-organisms in a sample comprising testing for an arabinopyranoside substrate cleaving activity of said microorganisms.
  • arabinopyranoside substrate cleaving activity refers to an activity cleaving the glycosidic bond between the arabinopyranose and its aglycone in said arabinopyranoside substrate.
  • An alternative for the expression “arabinopyranoside substrate cleaving activity” is the term “arabinopyranosidase”.
  • arabinopyranoside substrates are for instance, but not limited to the following: 4- methylumbelliferyl- ⁇ -L-arabinopyranoside, 4-trifluoromethylumbelliferyl- ⁇ -L- arabinopyranoside, o-nitrophenyl- ⁇ -L-arabinopyranoside, p-nitrophenyl- ⁇ -L- arabinopyranoside, indoxyl- ⁇ -L-arabinopyranoside and its chloro- and/or bromosubstituted derivatives (such as 5-bromo-4-chloro-3-indolyl-cc-L-arabinopyranoside, 5-bromo-6-chloro-3- indolyl- ⁇ -L-arabinopyranoside and 6-chloro-3-indolyl- ⁇ -L-arabinopyranoside), N- methylindolyl- ⁇ -L-arabinopyranoside, chlorophenol red ⁇ -L
  • the present invention relates to a method for detecting a fungal species in a sample comprising testing for an arabinopyranoside substrate cleaving activity of said fungal species.
  • the terms "fungal species” or “fungus” are interchangeable.
  • the present inventors were able for the first time to demonstrate a specific arabinopyranoside substrate cleaving activity in Aspergillus fumigatus which was not found in other fungi nor in other Aspergillus spp. tested. Therefore, in a further preferred embodiment, the present invention relates to a method for detecting Aspergillus fumigatus in a sample comprising testing for an arabinopyranoside substrate cleaving activity.
  • sample preferably is a "biological sample” and refers to any liquid or liquefied sample comprising bronchoalveolar liquid, blood, CSF, serum, urine, oropharyngeal exudate and swabs, tube culture, nose swabs, ear swabs and sputum.
  • samples suspected to contain microorganisms and more preferably fungal species can be analysed by the methods of the invention.
  • the present invention relates to a method as described above wherein said testing of arabinopyranoside substrate cleaving activity comprises the use of a fluorogenic, chromogenic or chemiluminogenic substrate. Examples of possible substrates for use in any of the methods of the invention are described above.
  • fluorogenic substrate refers to a compound which itself is non-fluorescent but which contains a structural part, i.e. the so-called fluorescent product, that emits light when liberated from the parent compound and photochemically excited.
  • chromogenic substrate refers to a compound which itself is not coloured but which contains a structural part, the so-called coloured product, that is or becomes coloured when liberated from the parent compound.
  • chemiluminogenic substrate refers to a compound which itself is not chemiluminescent but which contains a structural part, i.e. the so-called chemiluminescent product, that emits light when liberated from the parent compound and chemically excited.
  • the present invention relates to the use of 4-methylumbelliferyl- ⁇ -
  • L-arabinopyranoside which is a fluorogenic substrate.
  • the present invention relates to a method for the detection of micro-organisms, preferably fungal species in a sample comprising at least the following steps: a) concentrating the micro-organisms, preferably fungal species in said sample on a membrane filter, b) incubating the membrane filter of step a) on a growth medium to form microcolonies, c) removing said membrane filter from the growth medium, d) contacting said membrane filter with a solution comprising an arabinopyranoside substrate possibly supplemented with a membrane permeabilizer, e) possibly amplifying the signal by spraying said membrane filter with sodium hydroxide or another signal amplifying material, and, f) identifying the presence of a signal indicative for positive microcolonies on said membrane filter.
  • the present invention relates to a method for the detection of A. fumigatus in a sample comprising at least the following steps: a) concentrating the micro-organisms, including A. fumigatus, in said sample on a membrane filter, b) incubating the membrane filter of step a) on a growth medium to form microcolonies, c) removing said membrane filter from the growth medium, d) contacting said membrane filter with a solution comprising a solution of 4 methylbelliferyl- -L-arabinopyranoside possibly supplemented with a membrane permeabilizer, e) identifying the presence of a signal indicative for positive microcolonies on said membrane filter, and f) possibly amplifying said signal by spraying said membrane filter with sodium hydroxide or another signal amplifying material.
  • membrane permeabilizer refers to any compound capable of disrupting the cytoplasmic membrane of the micro-organism in order to facilitate the uptake of chemicals.
  • the membrane permeabilizers used in the methods of the invention are chosen from the group consisting of digitonine, toluene, amphotericin B, sodium lauryl sarcosinate, chloroform, dimethylsulfoxide, lysolecithin and higher alcohols, e.g. amyl alcohol.
  • a most preferred membrane permeabilizer used in the methods of the invention is digitonin
  • the membrane filter according to any of the methods of the invention can be chosen from nylon, polyester, polcarbonate, PVDF, cellulose- nitrate or any suitable membrane known in the art which can be combined with any of the methods described in the invention.
  • the membrane filter used in any of the methods of the invention is a nylon membrane filter.
  • signal amplifying material refers to a (chemical) compound that intensifies the signal indicative for the presence of positive colonies, for instance a compound that intensifies the light emission in fluorescence or chemiluminescence. It should be clear that the signal amplifying material also could be applied to the membrane (step f) before identification of monitoring any positive microcolony (step e).
  • signal indicative for positive microcolonies refers to microcolonies which are fluorescent, coloured or which emit light depending on the arabinopyranoside substrate used.
  • identification of positive microcolonies can be performed in different ways and with different means.
  • blue fluorescent microcolonies can be visualized under a 366 nm UV-lamp and inspected by the naked eye.
  • the expression "labelling with” means contacting of the microfilm containing the microcolonies with a solution comprising a labelled arabinopyranoside substrate.
  • the arabinopyranoside substrate is converted by the microorganisms or fungi into a light-emitting (or colored) product.
  • labelled arabinopyranside substrates are for instance 4-methylumbelliferyl- ⁇ -L- arabinopyranoside, 4-trifluoromethylumbelliferyl-o-L-arabinopyranoside, o-nitrophenyl- ⁇ -L- arabinopyranoside, p-nitrophenyl- ⁇ -L-arabinopyranoside, indoxyl- ⁇ -L-arabinopyranoside and its chloro- and/or bromosubstituted derivatives (such as 5-bromo-4-chloro-3-indolyl- ⁇ -L- arabinopyranoside, 5-bromo-6-chloro-3-indolyl- ⁇ -L-arabinopyranoside and 6-chloro-3-indolyl- ⁇ -L-arabinopyranoside), N-methylindolyl- ⁇ -L-arabinopyranoside, chlorophenol red ⁇ -L- arabinopyranoside,
  • the sensitivity of the detection can be enhanced, i.e. to detect small or weakly fluorescent microcolonies sooner than would be possible with the naked eye, by the use of an instrumental device.
  • an instrumental device consists of a light source (UV and visible light lamps), filters for the selection of excitation and emission wavelengths and a stereomicroscope. Through the use of appropriate filters, a variety of enzyme substrates yielding blue, green or red fluorescence can be used.
  • Preferred substrates for use in combination with the modular imaginer are 4-methylumbelliferyl- ⁇ -L- arabinopyranoside, resorufin- ⁇ -L-arabinopyranoside or N-methylindolyl- ⁇ -L- arabinopyranoside.
  • fluorescent single cells can be detected, without the need to form microcolonies, using the ChemScanTM (Chemunex, Ivry-sur-Seine, France) an apparatus developed for solid phase cytometry.
  • ChemScanTM ChemScanTM (Chemunex, Ivry-sur-Seine, France) an apparatus developed for solid phase cytometry.
  • fluorescently labelled cells are detected on a black membrane filter by a 488 nm laser beam, which scans the filter in 3 min.
  • Three photomultiplier tubes with wavelength windows set for the green (500-530 nm), amber (540-570 nm) and red (570-585 nm) regions of the emission spectrum of fluorescein detect the light emitted by the labelled cells.
  • the signals produced are processed by a computer using a series of software discriminants that enable the instrument to differentiate between labelled cells and background (e.g.
  • Scan results are displayed as colored spots on a membrane filter image in a primary scan map and, following elimination of background by software discrimination, secondary scan map.
  • the results displayed in the secondary map on the computer screen can be visually confirmed using an epifluorescence microscope with a moving stage, connected to the ChemScanTM.
  • the stage is driven by the ChemScanTM user interface.
  • the membrane filter in its holder is placed under the microscope, aligning it in exactly the same way as it is positioned in the ChemScanTM.
  • Highlighting of each fluorescent spot in the secondary scan map directs the motorized stage to the corresponding position on the membrane filter for inspection of the appearance of each detected fluorescent event.
  • Fluorescently labelled microorganisms in the above method can be obtained by incubation of the membrane with green-fluorescent arabinopyranosidase substrates such as fluorescein- ⁇ - L-arabinopyranoside, carboxyfluorescein- ⁇ -L-arabinopyranoside or N-methylindoxyl- ⁇ -L- arabinopyranoside for direct detection and/or visualisation of positive cells on the scan map of the ChemScanTM.
  • green-fluorescent arabinopyranosidase substrates such as fluorescein- ⁇ - L-arabinopyranoside, carboxyfluorescein- ⁇ -L-arabinopyranoside or N-methylindoxyl- ⁇ -L- arabinopyranoside for direct detection and/or visualisation of positive cells on the scan map of the ChemScanTM.
  • the present invention also relates to a method for the detection of fungal species, more preferably A. fumigatus, in a sample comprising at least the following steps: a) concentrating the micro-organisms in said sample on a membrane filter, b) contacting said membrane filter with a solution comprising a green-fluorescent arabinopyranoside substrate, c) identifying the presence of a signal indicative for positive microcolonies on said membrane filter using a ChemScanTM .
  • the membrane filters can be incubated with FITC-labelled (green- fluorescence) antibodies against the micro-organism to be detected (which is visualised the computer screen of the ChemScanTM) and a blue or red fluorescent arabinopyranosidase substrate, such as resorufin- ⁇ -L-arabinopyranoside (for visual microscopic confirmation of positive spots indicative for arabinosidase activity).
  • FITC-labelled antibodies against the micro-organism to be detected which is visualised the computer screen of the ChemScanTM
  • a blue or red fluorescent arabinopyranosidase substrate such as resorufin- ⁇ -L-arabinopyranoside (for visual microscopic confirmation of positive spots indicative for arabinosidase activity).
  • the present invention also relates to a method for the detection of fungal species, more preferably A. fumigatus, in a sample comprising at least the following steps: a) concentrating the micro-organisms in said sample on a membrane filter, b) contacting said membrane filter with a solution comprising a FITC-labelled anti A.fumigatus antibody and a green- or blue-fluorescent labelled arabinopyranoside substrate, c) identifying the presence of a signal indicative for positive microcolonies on said membrane filter using a ChemScanTM .
  • ChemScanTM and subsequently as green- or blue-fluorescent spots by visual confirmation of said positive spots under the epifluorescence microscope connected with the ChemScanTM, said green-or blue-fluorescence being indicative for arabinopyranosidase activity.
  • Infection with A. fumigatus results in a life-threatening disease. Therefore, the methods disclosed in the present invention and preferably in combination with any of the above described devices may have very important and sometimes vital (cardinal) advantages in reducing the detection time for A.
  • the invention also relates to a method for the detection of micro- organisms, preferably fungal species in a sample comprising at least the following steps: a) concentrating the micro-organisms including fungal species in said sample on a membrane filter, b) incubating the membrane filter of step a) on a growth medium to form microcolonies, said growth medium containing an arabinopyranoside substrate. c) identifying the presence of arabinopyranosidase positive microcolonies on said membrane filter.
  • a preferred micro-organism to be detected in any of the above methods is A.fumigatus.
  • the incubation step b) in any method of the invention may be performed at 45°C.This higher incubation temperature further increases the selectivity of the method(s) for detecting the presence of Aspergillus fumigatus.
  • the invention relates to any of the methods as described above for the detection and identification of Aspergillus fumigatus using 4-methylumbelliferyl- ⁇ -L-arabinopyranoside as a substrate for demonstrating the arabinopyranoside substrate cleaving activity of this species.
  • the invention also relates to a growth medium for use in any of the methods of the invention characterized in that it comprises Sabouraud glucose agar further comprising growth inhibitors for bacteria such as ticarcillin and clavulanic acid and further enriched by the addition of a mixture of growth factors, for example pyridoxine, thiamine, nicotinic acid, riboflavine, panthothenate, para-aminobenzoic acid (PABA) and inositol.
  • a growth medium for use in any of the methods of the invention characterized in that it comprises Sabouraud glucose agar further comprising growth inhibitors for bacteria such as ticarcillin and clavulanic acid and further enriched by the addition of a mixture of growth factors, for example pyridoxine, thiamine, nicotinic acid, riboflavine, panthothenate, para-aminobenzoic acid (PABA) and inositol.
  • PABA para-aminobenz
  • the present invention relates to a kit for diagnosing the presence of a micro-organism, preferably a fungal species in a biological sample comprising an arabinopyranoside substrate.
  • this kit comprises at least the following components: a) a membrane filter, b) possibly a growth medium for the fungal species to be detected, most preferably the growth medium as defined above, c) a fluorogenic, chromogenic or chemiluminogenic arabinopyranosidase substrate for testing the arabinopyranoside substrate cleaving activity, d) and possibly also sodium hydroxide or another signal amplifying material for spraying the membrane filter to amplify the signal.
  • the membrane filter referred to in a) preferably is a nylon membrane filter but can be any of the filters defined earlier.
  • the kit for diagnosing the presence of a micro-organism, preferably a fungal species in a sample comprising an arabinopyranoside substrate may further include an inducer for the arabinopyranoside substrate cleaving activity and/or a membrane permeabilize, said membrane permeabilizer can be chosen from the group already defined before.
  • the term "inducer” refers to molecules that promote the biosynthesis of an enzyme by enhancing the expression of the genes involved.
  • Table 1 Cleavage of 4- methylumbelliferyl-glucosides and esters by medically important fungi.
  • Table 2 Frequency table representing numbers of identified species comparing the arabinosidase based method and a conventional identification method.
  • strains were clinical isolates originating from the department of Clinical Biology, Bacteriology and Virology of the University Hospital of Ghent, Ghent, Belgium. Isolates were subcultured on Sabouraud glucose agar (SGA) (Difco Laboratories, Detroit, Mich.) and were incubated for at least 72 h at 37°C.
  • SGA Sabouraud glucose agar
  • Nylon membrane filters 47-mm diameter, 0.45 ⁇ m pore size (Gelman Sciences, Ann Arbor, Mich.) were used for filtration of the samples.
  • Other filters including nitrocellulose, polyamide, polysulfone, mixed cellulose esters and polytetrafluoroethylene filters were obtained from Millipore Corp. (Bedford, Mass.).
  • Absorbent fiberglass pads were from Gelman Sciences.
  • Sabouraud glucose agar was either supplemented with a combination of 2,500 ⁇ g/ml ticarcillin (2,344 ⁇ g/ml) -clavulanic acid (156 ⁇ g/ml) (Timentin®) (SmithKline Beecharn Pharma, Genval, Belgium) to yield SGA-T, with a mixture of pyridoxine (10 ppm), thiamine (1.0 ppm), nicotinic acid (0.1 ppm), riboflavine (10 ppm), panthothenate (1.0 ppm), PABA (1.0 ppm), and inositol (10 ppm) (all from Sigma Chemical Co., St.
  • Example 2 Method with visual detection of fluorescent microcolonies Screening for enzymatic activities.
  • the following 4-methylumbelliferyl (4-MU) derivatives were tested as enzyme substrates: acetate, N-acetyl- ⁇ -D-galactosaminide, ⁇ -L-arabinofuranoside, ⁇ -L-arabinopyranoside, ⁇ - D,D'-diacetylchitobioside, ⁇ -D-galactoside, ⁇ -D-glucoside, ⁇ -D-glucuronic acid, heptanoate, ⁇ - L-iduronide, ⁇ -D-lactoside, laurate, oleate, palmitate, phosphate, pyrophosphate and ⁇ -L- rhamnopyranoside. They were purchased from Sigma and Melford Laboratories (Ipswich, UK).
  • a 0.1 to 1 mg quantity of each substrate was dissolved in 1 ml of dimethylsulfoxide (Fluka, Buchs, Switserland), except 4-MU-palmitate and 4-MU-oleate, which were dissolved in dimethylformamide (Merck, Darmstadt, Germany).
  • Stock solutions were diluted in 0.1 M phosphate buffer (pH 4.5) and sterilized by filtration over Nalgene disposable units (0.45 ⁇ m pore size, 250 ml) (Nalge Co., Rochester, N.Y.). The solutions were dispensed in test tubes and frozen at -20°C until use. Procedure.
  • the filters were removed and placed on an absorbent fiberglass pad, impregnated with 340 ⁇ l of a buffered solution containing 0.1 % (w/v) 4-MU- ⁇ -L- arabinopyranoside, 0.1% (w/v) digitonin, acting as a membrane permeabilizer and 1 mM MgCl2, acting possibly as a co-factor for the enzyme.
  • a buffered solution containing 0.1 % (w/v) 4-MU- ⁇ -L- arabinopyranoside, 0.1% (w/v) digitonin, acting as a membrane permeabilizer and 1 mM MgCl2, acting possibly as a co-factor for the enzyme.
  • the filters were sprayed with 1.2 M sodium hydroxide and inspected under a 366 nm UV lamp. Blue fluorescent microcolonies indicated a positive reaction.
  • the membrane was reincubated at 45°C on the original SGA * -T medium for at least 3 days and the mycelial growth was examined macroscopically and microscopically after lactophenol cotton-blue staining.
  • part of the original sample was also filtered over a second nylon membrane. Inoculation was performed at 37°C for 3 days on SGA+ and the typical fungal colonies were characterized by microscopy.
  • Example 2 The procedure described in "Example 2" was used except for the final detection of the fluorescent microcolonies, which was carried out using a modular imager.
  • the latter consists of a light source, appropriate filters for selection of the excitation and emission wavelengths and a stereomicroscope.
  • the membrane filter is placed under the microscope and the fluorescent microcolonies are observed.
  • Useful enzyme substrates include 4-MU-a-L- arabinopyranoside (blue fluorescence), N-methylindoxyl-a-L-arabinopyranoside (green fluorescence) and resorufin-a-L-arabinopyranoside (red fluorescence).
  • Enzyme activities in A. fumigatus and other fungal species were assessed from their ability to cleave a series of 4-MU glycosides and esters.
  • the sensitivity of a given enzymatic reaction had to exceed 95%, i.e., less than 5% of strains of the target organism should be negative.
  • the specificity had to be ⁇ 95%, i.e., the enzyme should occur in ⁇ 5% of other fungal species.
  • the rate of microcolony formation on the nylon membrane filter depended on the composition of the growth medium and the temperature, but was also influenced by the cellular stage of growth initially present in the sample (mycelium or spores) and the administration of antimycotics to the patient before sampling.
  • the growth promoting activity of the various media was compared in broth on the basis of turbidity measurements as a function of time.
  • the enriched SGA * yielded faster growth than SGA, CMT, PGA and CDA (results not shown).
  • a combination of ticarcillin-clavulanic acid has previously proven effective to this end (Bauters et al., 1999).
  • To ensure that antibacterials had no negative effect on the proliferation of A. fumigatus mycelial growth was monitored in broth for 44 h by turbidimetry. To this end, an amount of 10 2 conidia of three A. fumigatus strains were inoculated in 10 ml SGA * and SGA * - T broth and incubated (results not shown). No differences in growth rate were observed in both media. Growth at 45°C was superior to that at 37°C and entails additional selectivity towards bacteria and yeasts.
  • Hyphae started to grow and differentiate after an average time of 10 (MF) and 14 h (no MF), vesicles were observed after 14 and 17 h, while the end-point of the growth phase, i.e., the presence of phialides and conidia, was observed after 17 and 23 h, respectively.
  • Microcolony formation was strongly affected by the developmental stage of the fungus in the sample. When starting from a sample containing mycelium, as in sputum or bronchoalveolar liquid, microcolonies appeared after approximately 11-14 hours. However, in case of spores the detection time was extended to at least 24 hours. Delayed growth was also observed in samples from patients treated with amphotericin B or itraconazole.
  • the first fluorescent microcolonies on the membrane filter could be visualized after approximately 17 hours.
  • the membrane filter after microcolony formation, is removed from the SGA * -T and placed on an absorbent pad containing the substrate and 0.1% digitonin as a membrane permeabilizer, a further reduction of three hours in detection time was obtained.
  • the method has been applied to 188 clinical specimens, both positive and negative for A. fumigatus.
  • the results with reference to a conventional plate isolation method with microscopic confirmation are listed in Table 2. Sensitivity and specificity were 96.9 and 100%, while the positive and negative predictive values were 100 and 95.8%, respectively. The overall efficiency of the method was 98.2%.
  • Samples after appropriate pretreatment as specified in Example 2, are filtered over a 25-mm black polyester membrane filter (Cycloblack') with a pore size of 2.0 ⁇ m. After filtration, the membrane filter is placed on a buffered solution containing a substrate for arabinopyranosidase which emits in the green region of the spectrum and incubated at 30°C or 37°C for variable times (ranging from 20 min to 3 hours: e.g. 30 min, 1 h, 2h). To this end, fluorescein-a-L-arabinopyranoside, carboxyfluorescein-a-L-arabinopyranoside and N- methylindoxyl-a-L-arabinopyranoside can be used.
  • the membrane filter is then inserted in a holder, placed in a solid phase cytometer (ChemScan , Chemunex, Ivry-sur-Seine, France) and scanned by a laser beam for 3 minutes.
  • Green fluorescent cells indicate arabinopyranosidase activity, typical for Aspergillus fumigatus.
  • the membrane filter is placed on a buffered solution containing FITC-labelled antibodies against Aspergillus fumigatus and a substrate for arabinopyranosidase, e.g. resorufin-a-L-arabinopyranoside and incubated at 30°C or 37°C for variable times.
  • the membrane filter is then inserted in a holder, placed in a solid phase cytometer
  • A. fumigatus 3 (n 48) 100 98 100 100 100 100 100 94 100 100
  • A. clavatus (n 4) 0 0 0 100 100 100 100 100 100 100 100 100 100 100 100 100 100
  • A. terreus (n 12) 0 0 8 100 100 0 100 100 100 100 100
  • A. versicolor (n 6) 0 0 0 100 100 0 100 100 0 100
  • F. oxysporum (n 4) 100 0 0 100 100 100 100 100 50 100
  • A. corymbifera (n 2) 100 0 0 100 100 0 100 100 100 100 100

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  • Genetics & Genomics (AREA)
  • Toxicology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne la détection de micro-organismes et leur identification sur la base d'une activité enzymatique spécifique présente dans ledit micro-organisme à détecter. L'invention concerne également des méthodes et des kits permettant de détecter les micro-organismes, et de préférence des espèces fongiques, à l'aide d'un substrat arabinopyranoside. Elle concerne plus particulièrement des méthodes et des kits permettant de détecter un Aspergillus fumigatus de manière rapide et sensible.
PCT/EP2001/002727 2000-03-10 2001-03-12 Procede rapide et sensible permettant de detecter un aspergillus fumigatus WO2001066790A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2001240692A AU2001240692A1 (en) 2000-03-10 2001-03-12 Rapid and sensitive plate method for detection of aspergillus fumigatus

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
EP00870041A EP1132481A1 (fr) 2000-03-10 2000-03-10 Détection d'Aspergillus fumigatus
EP00870041.1 2000-03-10
US19482300P 2000-04-05 2000-04-05
US60/194,823 2000-04-05

Publications (2)

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WO2001066790A2 true WO2001066790A2 (fr) 2001-09-13
WO2001066790A3 WO2001066790A3 (fr) 2002-03-28

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WO (1) WO2001066790A2 (fr)

Cited By (5)

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Publication number Priority date Publication date Assignee Title
EP1567661A2 (fr) * 2002-11-26 2005-08-31 Expressive Constructs, Inc. Procedes, biocapteurs et necessaires de detection et d'identification de champignons
GB2472429A (en) * 2009-08-06 2011-02-09 Munters Ltd A method of restoring a water-damaged vehicle
FR2970974A1 (fr) * 2011-02-01 2012-08-03 Biomerieux Sa Milieu de culture de microorganismes comprenant l'acide para-aminobenzoique comme agent selectif
US9017963B2 (en) 2002-01-31 2015-04-28 Woundchek Laboratories (Us), Inc. Method for detecting microorganisms
CN109988855A (zh) * 2017-12-29 2019-07-09 博奥生物集团有限公司 用于检测六种曲霉的lamp引物组合及其应用

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9017963B2 (en) 2002-01-31 2015-04-28 Woundchek Laboratories (Us), Inc. Method for detecting microorganisms
EP1567661A2 (fr) * 2002-11-26 2005-08-31 Expressive Constructs, Inc. Procedes, biocapteurs et necessaires de detection et d'identification de champignons
EP1567661A4 (fr) * 2002-11-26 2007-01-10 Expressive Constructs Inc Procedes, biocapteurs et necessaires de detection et d'identification de champignons
GB2472429A (en) * 2009-08-06 2011-02-09 Munters Ltd A method of restoring a water-damaged vehicle
GB2472429B (en) * 2009-08-06 2015-04-22 R3 Polygon Uk Ltd Restoration techniques
FR2970974A1 (fr) * 2011-02-01 2012-08-03 Biomerieux Sa Milieu de culture de microorganismes comprenant l'acide para-aminobenzoique comme agent selectif
WO2012104544A1 (fr) * 2011-02-01 2012-08-09 bioMérieux Milieu de culture de microorganismes comprenant l'acide para-aminobenzoique comme agent selectif
US10233477B2 (en) 2011-02-01 2019-03-19 Biomerieux Culture medium for microorganisms including para-aminobenzoic acid as a selective agent
CN109988855A (zh) * 2017-12-29 2019-07-09 博奥生物集团有限公司 用于检测六种曲霉的lamp引物组合及其应用
CN109988855B (zh) * 2017-12-29 2022-07-12 博奥生物集团有限公司 用于检测六种曲霉的lamp引物组合及其应用

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AU2001240692A1 (en) 2001-09-17
WO2001066790A3 (fr) 2002-03-28

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