HUE035216T2 - Eljárás aspergilus fertõzés kockázatának meghatározására mikrobiális illékony szerves vegyületek kimutatására alapozva - Google Patents

Description

METHOD FOR DFTERMiNiNG A RISK QE ASPERGILLUS CONTAMINATION BASED ON THE DETECTION OE MiCROBiAL VOLATILE ORGANIC COMPONENTS

Technical field of the invention [0001] The present invention relst.es to a method for determining a chemical fingerprint specific to an Aspergillus contamination in indoor environments.

[0002} Here, "indoor environment" shall mean a confined space inside a building which is ventiiated in a non-continuous manner. Examples of indoor environments can be found in residences, museums, churches, cellars, historic monuments, administrative buildings, schools and hospitals.

[0003] The fungai growth is accompanied by emission of MVOC [Microbial Volatile Organic Components) from the start of growth and during ail the growth phases of the micromycetes.

Prior art [0004] Application WO 2004/051226 is known in this field and relates to methods for monitoring at-risk environments for the presence or absence of microbes. This invention is characterised by the search fora cnpGO marker, [0005} However, this document is limited to a search for one chaperonin [cnp60], which implies cellular extractions which must be selective for this chaperonin, [00061 This type of method implies significant growth of micromycetes, followed by collection and extraction of the chaperonin. it is of course preferable to avoid the generation of micromycetes, which are potentially dangerous to human beings, in addition, detecting chaperonin is very difficult in comparison to detecting VOCs.

[0007} To overcome these disadvantages, the applicant has developed a method for detecting a fungal contamination of an indoor environment, using a calculation of a chemicai fungaheontamination index. This method is described in application WO 2008/125770. This tool made it possible to determine the presence of micromycetes in 37 to 42% of French homes during a national campaign of the Observato/re tie /o Quo/ffe tie ΓΑΐί mtef/eui' [Mouiarat et ai., 2000a).

[0008] However this method does not make it possible to specifically conclude whether or not there is a risk of Aspergillus contamination. (0009] Polizzi et ai, (Fungal Biology, IIS, 941-953,. 2012} describe the detection of different MVOC in rooms contaminated by micromycetes, and the comparison of the detected MVOCs with the MVOC profiles produced by micromycetes isolated from these same rooms, which included several Aspergillus species, when they are cultivated in vitro on MBA medium (Malt extract agar}. The authors report that 4 MVOC {«-copaene, geranyl acetone, trans-caiamenene, and α-calacorene}, detected in the rooms where the Aspergillus presence was predominant, are also produced in vitro by certain strains of the species Asper p i!lu s us t us.

Description of the invention [0010] in this context, the fungal contamination index developed by the applicant and described in application WO 200S/125770 could he supplemented and refined for early detection of an Aspergillus growth, [0011] In order to overcome the disadvantages of the prior art, the applicant proposes a method for determining an Aspergillus contamination risk in an indoor environment, comprising the following steps; (a) taking a sample of air in the indoor environment, then (b) detecting MVOCs in the sample, this search comprising a search for a chemical fingerprint comprising st least one target molecule which is an MVOC, associated with an Aspergillus metabolism, said target molecule being selected from the following MVOCs; 1,4-pentadiene, 4-heptanone, dimethyl disulfide, methoxybenzene, 1,3-hut.anedioi, 1,4-hexadiene, l~methoxy-2- methylbenzene, l-octen-B-one, 1-pentene, 2(5H)-Furanone, 2-methyl-isoborneoi, 3,3-dichioro-l-propene, 3-butyn-l-ol, 3-heptanol, 3-heptanone, 3-methyl-2-bntano!, 3-mefhyihexane, 4~heptanoi, a-methyi-S-hexanone, caryophyllene, dimethyl trisulfide, eremophilene, germacrene D, isoiedene, Iongifoiene, methyl-2- ethyl bexanoate, muurolane and terpinoiene, [0012] Here "risk of Aspergillus contamination" shall mean a development of micromycetes of the genus Aspergiiius on a given medium, [0013] In a particularly advantageous manner, the detection of such target molecules is easier and quicker than the detection of Aspergiiius strains or soluble Aspergillus metabolites.

[0014] According to 3 preferred embodiment, step (b) comprises the following sub-steps before the search for a chemical fingerprint comprising at ieast one target molecule which is an MVOC associated with Aspergillus metabolism: - detection of VOCs in the sample, which includes detection of the presence or absence of certain predetermined VOCs coming from fungal metabolism, said predetermined VOCs comprising at least one VOC from each of the following three VOC categories: (1) VOCs which are emitted independently of the fungal species and the medium thereof, and which are only emitted by fungal species; (2) VOCs which are emitted independently of the fungal species and the medium, and which are emitted by non-fungai biological species; (3) VOCS which are emitted as a function of the fungal species and/or the medium thereof; ·· calculation of a chemical fungal-contamination index as a function respectively of the presence and absence of predefined VOCs coming from the fungal metabolism.

[0015] Here, “medium" of a fungal species shall mean the material on which the fungal species grows, preferably a construction material such as wailpaper, glass fabric or other, [0016] Advantageously, the chemical fingerprint is specific to at least one Aspergillus species selected from Asperpn'/us restr/cfos, Aspergillus versicoior; Aspergillus sydowfr and Aspergillus niger.

[0017] Alternatively, said target molecule is selected from the group comprising: 1,4-pentadiene, 4-heptanone, dimethyl disulfide, methoxybenzene. These target molecules are specific to a plurality of Aspergillus species, [0018] According to another variant, said target molecule is selected from the group comprising; l,3~butanedics!, 1,4-hexadiene, l-methoxy-2-methyibenzene, l-octen- 3- one, 1-psnfene, 2(5H)-foranone, 2-methyi-isoborneoi, 3,3-dich!oro-l~ propene, 3-hutyml-oi, 3-heptanol, 3-beptanone, 3-metby!-2-butanoi, 3- methyihexane, 4-heptanol, 4- methyl-2 hexanone, caryophyllene, dimethyl trisulfide, eremophiiene, germacrene D, isoledene, ionglfoiene, methyl-2-ethylhexanoate, muurolane and terpinoiene, These target molecules are specific to one or two Aspergillus strains.

[0019] Hence, the method according to the invention can be carried out with one of said target molecules which is specific to one or two Aspergillus strains. Alternatively, this target molecule is specific to more than two Aspergillus species, [0020] According to a preferred variant, the chemical fingerprint comprises at least two target molecules, [002.11 According to another variant, the chemical fingerprint comprises all said target molecules.

[0022] Advantageously, the method comprises a step of searching for fungal contamination zones, carried out before step (a). Hence, the. method according to said variant starts with this step.

[0023] The method according to the second embodiment of the invention is particularly useful for early detection of an Aspergilius contamination risk, i,e. before the appearance of significant quantities for microbiological detection, This eariy detection possibility Is even more interesting since it does not. require a direct detection of Aspergilius species. The Aspergiiius contamination can thus be deduced at an eariy stage in the development of the fungi. Here "eariy-stage” shall mean growth, a stage where the micromycetes are invisible on the surface of the medium, and preferably undetectable by microbiological analysis of the air, but nevertheless produce inhaiable metabolites and degradation products and are responsible, in some cases, fondnesses.

Detaiied description of an embodiment [002.4] Tbe present invention Is based on a laboratory study of VQC emissions from 4 species of the genus Aspergilius:

Aspergiiius restrictus, A. versicoior, •A, sydow/y A. u/per.

[0025] These species have been cultivated away from Sight, at 25 *C, on various sterilised materials that are frequently found contaminated in indoor environments. A non -emitting reference medium, consisting of glass fibre soaked with a nutritive solution, was also used for ait the tested strains. The nutritive solution used is, for example, an aqueous solution comprising in particular K2WPO4, KCf, Mg5O4, PeS04, Glucose and MaNO3. This solution is buffered to a pH of 7.4,

St is of course possible to use another known nutritive solution without going beyond the scope of the invention. (0026) The handling plan is summarised in table 1 below.

Table 1: fungal strains tested as a function of tbe growth medium

(00271 in table 1, a cross indicates the identification of an Aspergillus strain (rows} which grows on the growth medium (columns). (002.8} The reference medium here is a positive control for confirming tbe capacity of the Aspergillus strain to grow. Tbe medium used was glass fibre soaked in a nutritive solution described above, Of course, another known reference medium can be used. (0029) The studied strains ail grow on the reference medium and on at least one other growth medium selected from those tested. The A. sydowii strain only grows on fiax, The other strains A, restrtctus, A, versicolor, A. sydowii and A. oiger grow on more than two growth media among those tested. (0030) During this study, 28 VOCs were identified uniquely from Aspergiilus strains (Table 2):

Table 2; Compounds emitted in the presence of Aspergillus growth

[00311 Tahie 2 lists the VOCs emitted in the presence of various Aspergillus strains. The samplings and the analysis of these VOCs were carried out after 7 days incubation of the strains at 25 °C, The crosses indicate identification of the VOCs from the growth of the species on at least one growth medium, (0032] As can be seen in table 2, the first four VOCs are markers for four Aspergillus species, while the other VOCs are markers for one or two Aspergillus species.

[0033] Hence this list of compounds can be split Into 2 groups: [0034] The compounds emitted by the group of Aspergillus species tested (group X highlighted in grey in the table). (0035) The compounds emitted by at least one and at most three of the four Aspergiiius species tested (group 2), (0036) From a practical point of view, after determining the presence of a fungal growth, for example by the fungal contamination index, the search for specific targets listed in table 2 makes it possible to warn of a probable development of Aspergillus species. Indeed, the presence of ai least one of said target species indicates the probable presence of a growth of Aspergillus species. (0037) The number of tracers identified is correlated with the probability of the presence of an Aspergiiius growth. The absence of compounds from group 1 reduces this probability. (0038) Other target molecules can be identified. In general, target molecules of this type can consist of any VOC linked with the Aspergiiius metabolic schemes, in other words a VOC produced by a strain of Aspergillus during Aspergillus metabolism. •0039) Hence, the determining of an Aspergiiius contamination fingerprint based on detection of specific chemical MVOCs enables the fungal contamination indices already developed in application WO 2008/125770 to be completed, by providing clear and reliable criteria on the decisions concerning, for example, occupation and renovation of contaminated buildings, (0040) In the implementation of the method according to a preferred variant, the following steps are performed: a) taking of an air sample in an indoor environment, for example close to zones suspected of being contaminated; b) detection of VGCs in the sample, which comprises detecting the presence or absence of certain predetermined VOCs arising from fungal metabolism, said predetermined of VOCs comprising at least one VOC from each of the three following VOC categories: (1) VOCs which are emitted independently of the fungal species and the medium thereof, and which are only emitted by f ungal species; (2) VOCs which are emitted Independently of the fungal species arid the medium, but which can also have other biological origins. Here VOCs having "other biological origins" shall mean in particular VOCs emitted by non-fungal biological species; (3} VQCs which are emitted as a function of the fungal species and/or the medium thereof; c) calculation of a chemical fungal-contamination index as a function respectively of the presence and absence of predefined VOGs arising from fungal metabolism, complying with the method described in application WO 2008/125770, in order to determine if there is a fungal contamination;

Then in order to determine if there is an Aspergillus contamination, the foiiowing steps are performed* d) search for a least one target molecule, which is a VOC coming from Aspergillus metabolism, in particular at least one target molecule selected from the group comprising: 1,4-pentadiene, 4-heptanone, dimethyl disulfide, methoxybenzene, 1.,3-butanediol, 1,4-bexadiene, l-methoxy-2- methylbenzene, l-octen-3-one, 1-pentene., 2{5H)-furanone, 2-methYi isoborneol, 3,3-dichloro-1.-propene, 3-but.yn-l-ol, 3-heptanol, 3-heptanone, 3-methyl-2-butanol, 3-methylhexane, 4-heptanol, 4-methyi-2-hexanone, caryophyliene, dimethyi trisulfide, eremophiiene, germacrene 0, isofedene, longifolene, met.hyl-2-ethyl hexanoate, muurolane, terpinolene; and preferably, e) search for a chemical fingerprint comprising at least two of said target molecules, [0041[ in an interesting, novel and inventive manner, the results from steps d) and ei make it possible to determine, with precision, clarity and reliability, whether or not there is a risk of Aspergillus contamination.

[0042] This embodiment, of course leads to more complete results than those of the prior art, in that it. concerns not only concluding that there is a fungal contamination without any visible sign of fungai growth, but also determining and Aspergillus growth in a precise and reliable manner, [0043] In another variant of the invention, it is also possible to search for fungal contamination zones, then take a sample of the air close to said fungal contamination zones before searching for said target molecule or molecules listed above.

[00441 Such a search for fungai contamination zones may be made for example by the naked eye, through microscopy analysis or by microbiological or biochemical tests.

[0045] The taking of the air sampie is carried out, for example, by diffusive sampling on a carbograph-4 type solid absorbent. The detection is performed, for example., by gas phase chromatography followed by mass spectrometry {GC/MS). Other detection methods can also be used.

Claims (8)

1. Szabadalmi igénypontok

   1. Eljárás aspergillus fertőzés kockázatának meghatározására belső környezetben, amely az alábbi lépéseket foglalja magában: a) levegő mintavétel a belső környezetben, majd b) a mintában a mikrobiális illékony szerves vegyületek (VOCm-ek) kimutatása, azzal jelle mezve, bog}··' a b) lépés tartalmaz legalább egy cél molekulára, amely egy aspergillus metabolizmushoz köthető VOCm, irányuló kémiai mintázat vizsgálatot, a nevezett célmolekulál a. következő VOCtn-ek közül választjuk: 1,4-pentadién, 4-heptanon, di~ metil-dÍszuífid, metoxi-benzoí, í ,3-butándiol, 1,4-hexadién, l-metoxi-2-metil--benzol, 1-oktén-3-on, Operáén, 2(511 ffnranon, 2-tnetil-izoborneol, 3J-diklór-l--propén, 3-butin-l-ol, 3~beptanol, 3-heptanon, 3-metil-2-butanol. 3-metil~hexán. 4--heptanok 4-metil-2-hexanon, kariofdlén, dlmetil-triszulfid, eremofilén, germakrén D, izoledén, longifoíén, metil-l-etíl-hexanoát, muurolán, ierpinolén.
2. 2. Az 1, igénypont szerinti eljárás aspergillus fertőzés kockázatának meghatározására belső környeze tben, azzal jellemezve, hogy a b) lépés az alábbi al- lépéseket tartalmazza a legalább egy célmolekulára, amely egy aspergillus metabolizmushoz köthető VOCm, irányuló kémiai mintázat vizsgálat előtt: - gomba metabolízmusből eredő néhány előre meghatározott, illékony szerves vegyület (VOC) jelenlétének vagy hiányának kimutatása, amely előre meghatározott VOC-ok az alábbi három VOC kategória mindegyikéből legalább egyet tartalmaznak: (1) a gomba tájtól és annak hordozójától függetlenül kibocsátott VOC-ok és amelyeket csak gomba fajok: bocsátanak ki; (2) a gomba fejtől és annak hordozójától függetlenül kibocsátott VOC-ok, és amelyek nem-gomba biológiai fejtól erednek; (3) a gomba iáitól és/vagy annak hordozójától függően kibocsátott VOC-ok; - a gombás fertőzés kémiai indexének kiszámítása a gomba metabolizmnsból eredő előre meghatározott VOC jelenlétének, illetve hiányának megfelelően.
3. 3. Az 1. vagy 2. igénypont szerinti eljárás, azzal jellemezve, hogy a kémiai mintázat specifikus legalább egy, az alábbiak közül választott, aspergillus fajra: zfsy.?erg///t«· mv-AíVtzr, .d.sy?ergü7w,s- verv/co/or, rtspergú’hí.s' sydowii. zlspargiZZuv níger.
4. 4. Az 1-3. igénypontok bármelyike szerinti eljárás, azzal jellemezve, hogy a kémiai mintázat legalább két nevezett célmolekulál tartalmaz,
5. 5. Az 1-4. igénypontok bármelyike szerinti eljárás, azzal jellemezve, hogy a kémiai mintázat az összes nevezett célmolekulát tartalmazza.
6. 6. Az 1-3. igénypontok bármelyike szerinti eljárás, azzal jellemezve, hogy a célmolekula egy vagy két Aspergillus törzsre specifikus. 7. A 6, igénypont szerinti eljárás, azzal jellemezve, hogy a célmolekulát az alábbiakat tartalmazó csoportból választjuk: 1,3-butándíol, 1,4-hexadién, l-metoxí-2-metil· -benzol, I-oktén-3-on, 1-petitén, 2(5H)-furanon, 2~metil“izöborneol, 3,3-diklór-l-propén, 3--butín-1-ol, 3-beptanol, 3-beptanon, 3~snetil-2-b«tanol, 3-metíl-hexán, 4-beptanol, 4~metil~2--hexánon, karioftllén, dtmetii-triszulfd, eremofdén, germakrén D, ízöledén, longífolén, metil-2-etíbhexanoát, mnurolán, terminolén.
7. 8. Az. 1-7. igénypontok bármelyike szerinti eljárás, azzal jellemezve, hogy a célmolekula több, mint kel AspergiUus törzsre specifikus, és előnyösen az alábbiakat tartalmazó csoportból választjuk: 1,4-pentadién, 4-heptanon, dímetíl-díszulfid, metoxí-benzol,
8. 9. Az 1-8, igénypontok bármelyike szerinti eljárás, amely gombás fertözöttségi zónák keresését tartalmazza az (a) lépés előtt.