DK2708604T3 - Fremgangsmåde til at bestemme risikoen for Aspergillus-kontaminering baseret på detektionen af flygtige organiske mikrobielle forbindelser - Google Patents

Description

METHOD FOR DETERMINING A RISK OF ASPERGILLUS CONTAMINATION BASED ON THE DETECTION OF MICROBIAL VOLATILE ORGANIC COMPONENTS

Technical field of the invention

The present invention relates to a method for determining a chemical fingerprint specific to an Aspergillus contamination in indoor environments.

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

The fungal growth is accompanied by emission of MVOC (Microbial Volatile Organic Components) from the start of growth and during all the growth phases of the micromycetes.

Prior art

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 for a cnp60 marker.

However, this document is limited to a search for one chaperonin (cnp60), which implies cellular extractions which must be selective for this chaperonin.

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.

To overcome these disadvantages, the applicant has developed a method for detecting a fungal contamination of an indoor environment, using a calculation of a chemical fungal-contamination 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 Observatoire de la Qualité de l'Air Intérieur (Moularat et al., 2008a).

However this method does not make it possible to specifically conclude whether or not there is a risk of Aspergillus contamination.

Polizzi et al. (Fungal Biology, 116, 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 MEA medium (Malt extract agar). The authors report that 4 MVOC (α-copaene, geranyl acetone, trans-calamenene, and a-calacorene), detected in the rooms where the Aspergillus presence was predominant, are also produced in vitro by certain strains of the species Aspergillus ustus.

Description of the invention

In this context, the fungal contamination index developed by the applicant and described in application WO 2008/125770 could be supplemented and refined for early detection of an Aspergillus growth.

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 at 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-butanediol, 1,4-hexadiene, 1-methoxy-2- methyl benzene, l-octen-3-one, 1-pentene, 2(5H)-furanone, 2-methyl-isoborneol, 3,3-dichloro-l-propene, 3-butyn-l-ol, 3-heptanol, 3-heptanone, 3-methyl-2-butanol, 3-methylhexane, 4-heptanol, 4-methyl-2-hexanone, caryophyllene, dimethyl trisulfide, eremophilene, germacrene D, isoledene, longifolene, methyl-2- ethyl hexanoate, muurolane and terpinolene.

Here "risk of Aspergillus contamination" shall mean a development of micromycetes of the genus Aspergillus on a given medium.

In a particularly advantageous manner, the detection of such target molecules is easier and quicker than the detection of Aspergillus strains or soluble Aspergillus metabolites.

According to a preferred embodiment, step (b) comprises the following sub-steps before the search for a chemical fingerprint comprising at least 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-fungal 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.

Here, "medium" of a fungal species shall mean the material on which the fungal species grows, preferably a construction material such as wallpaper, glass fabric or other.

Advantageously, the chemical fingerprint is specific to at least one Aspergillus species selected from Aspergillus restrictus, Aspergillus versicolor, Aspergillus sydowii and Aspergillus niger.

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.

According to another variant, said target molecule is selected from the group comprising: 1,3-butanediol, 1,4-hexadiene, l-methoxy-2-methylbenzene, l-octen-3-one, 1-pentene, 2(5H)-furanone, 2-methyl-isoborneol, 3,3-dichloro-l- propene, 3-butyn-l-ol, 3-heptanol, 3-heptanone, 3-methyl-2-butanol, 3- methylhexane, 4-heptanol, 4-methyl-2-hexanone, caryophyllene, dimethyl trisulfide, eremophilene, germacrene D, isoledene, longifolene, methyl-2-ethylhexanoate, muurolane and terpinolene. These target molecules are specific to one or two Aspergillus strains.

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.

According to a preferred variant, the chemical fingerprint comprises at least two target molecules.

According to another variant, the chemical fingerprint comprises all said target molecules.

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.

The method according to the second embodiment of the invention is particularly useful for early detection of an Aspergillus contamination risk, i.e. before the appearance of significant quantities for microbiological detection. This early detection possibility is even more interesting since it does not require a direct detection of Aspergillus species. The Aspergillus contamination can thus be deduced at an early stage in the development of the fungi. Here "early-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 inhalable metabolites and degradation products and are responsible, in some cases, for illnesses.

Detailed description of an embodiment

The present invention is based on a laboratory study of VOC emissions from 4 species of the genus Aspergillus: - Aspergillus restrictus, -A. versicolor, - A. sydowii, - A. niger.

These species have been cultivated away from light, 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 all the tested strains. The nutritive solution used is, for example, an aqueous solution comprising in particular K2HP04, KCI, MgS04, FeS04, Glucose and NaN03. This solution is buffered to a pH of 7.4.

It is of course possible to use another known nutritive solution without going beyond the scope of the invention.

The handling plan is summarised in table 1 below.

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

In table 1, a cross indicates the identification of an Aspergillus strain (rows) which grows on the growth medium (columns).

The reference medium here is a positive control for confirming the capacity of the Aspergillus strain to grow. The medium used was glass fibre soaked in a nutritive solution described above. Of course, another known reference medium can be used.

The studied strains all grow on the reference medium and on at least one other growth medium selected from those tested. The A. sydowii strain only grows on flax. The other strains A. restrictus, A. versicolor, A. sydowii and A. niger grow on more than two growth media among those tested.

During this study, 28 VOCs were identified uniquely from Aspergillus strains (Table 2):

Table 2: Compounds emitted in the presence of Aspergillus growth

Table 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.

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.

Hence this list of compounds can be split into 2 groups:

The compounds emitted by the group of Aspergillus species tested (group 1 highlighted in grey in the table).

The compounds emitted by at least one and at most three of the four Aspergillus species tested (group 2).

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 at least one of said target species indicates the probable presence of a growth of Aspergillus species.

The number of tracers identified is correlated with the probability of the presence of an Aspergillus growth. The absence of compounds from group 1 reduces this probability.

Other target molecules can be identified. In general, target molecules of this type can consist of any VOC linked with the Aspergillus metabolic schemes, in other words a VOC produced by a strain of Aspergillus during Aspergillus metabolism.

Hence, the determining of an Aspergillus 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.

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 VOCs 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 fungal species; (2) VOCs which are emitted independently of the fungal species and 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) VOCs 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 VOCs 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 following 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-hexadiene, 1-methoxy-2- methylbenzene, l-octen-3-one, 1-pentene, 2(5H)-furanone, 2-methyl-isoborneol, 3,3-dichloro-l-propene, 3-butyn-l-ol, 3-heptanol, 3-heptanone, 3-methyl-2-butanol, 3-methylhexane, 4-heptanol, 4-methyl-2-hexanone, caryophyllene, dimethyl trisulfide, eremophilene, germacrene D, isoledene, longifolene, methyl-2-ethyl hexanoate, muurolane, terpinolene; and preferably, e) search for a chemical fingerprint comprising at least two of said target molecules.

In an interesting, novel and inventive manner, the results from steps d) and e) make it possible to determine, with precision, clarity and reliability, whether or not there is a risk of Aspergillus contamination.

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 fungal growth, but also determining and Aspergillus growth in a precise and reliable manner.

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.

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

The taking of the air sample 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 (9)

1. Fremgangsmåde til at bestemme risikoen for Aspergillus-kontaminering i et indendørs miljø omfattende de følgende trin: a) at indsamle en prøve af luften i det indendørs miljø, og b) at detektere flygtige organiske mikrobielle forbindelser (MVOC) i prøven, kendetegnet ved at trin b) omfatter søgning efter et kemisk fingeraftryk omfattende mindst et målmolekyle, der er et MVOC associeret med Aspergillus-metabolisme, hvilket målmolekyle vælges blandt de følgende MVOC'er: 1,4-pentadien, 4-heptanon, dimethyldisulfid, Methoxybenzen, 1,3-butanediol, 1,4-hexadien, l-methoxy-2-methyl-benzen, l-octen-3-on, 1-penten, 2(5H)-furanon, 2-methyl-isoborneol, 3,3-dichlor-l-propen, 3-butyn-l-ol, 3-heptanol, 3-heptanon, 3-methyl-2-butanol, 3-methylhexan, 4-heptanol, 4-methyl-2-hexanon, caryophyllen, dimethyltrisulfid, eremophilen, Germacren D, isoleden, longifolen, methyl-2-ethylhexanoat, muurolan, terpinolen.

2. Fremgangsmåde til at bestemme risikoen for Aspergillus-forurening i et indendørs miljø ifølge krav 1, kendetegnet ved at trin b) omfatter de følgende undertrin før søgningen efter et kemisk fingeraftryk omfattende mindst et målmolekyle, der er et MVOC associeret med Aspergillus-metabolisme: - at detektere tilstedeværelsen af eller manglen på bestemte forudbestemte flygtige organiske stoffer (VOC) stammende fra svampemetabolisme, idet disse forudbestemte VOC'er omfatter mindst et VOC fra hver af de følgende tre kategorier af VOC'er: (1) VOC'er, der frigives uafhængigt af svampearten og dens bærer og kun frigives af svampearter; (2) VOC'er, der frigives uafhængigt af svampearten og bæreren, og som frigives af biologiske ikke-svampearter; (3) VOC'er, der frigives afhængigt af svampearten og/eller dens bærer - at beregne et kemisk indeks for svampekontaminering som funktion af henholdsvis tilstedeværelsen afog manglen på foruddefinerede VOC'er frigiver ved svampemetabolisme.

3. Fremgangsmåde ifølge krav 1 eller 2, kendetegnet ved at det kemiske fingeraftryk er specifikt for mindst en Aspergillus-art valgt blandt Aspergillus restrictus, Aspergillus versicolor, Aspergillus sydowii, Aspergillus nlger.

4. Fremgangsmåde ifølge et af kravene 1 til 3, kendetegnet ved at det kemiske fingeraftryk omfatter mindst to af målmolekylerne.

5. Fremgangsmåde ifølge et af kravene 1 til 4, kendetegnet ved at det kemiske fingeraftryk omfatter alle målmolekylerne.

6. Fremgangsmåde ifølge et af kravene 1 til 5, kendetegnet ved at målmolekylet er specifikt for en eller to Aspergillus-stammer.

7. Fremgangsmåde ifølge krav 6, kendetegnet ved at målmolekylet vælges fra gruppen omfattende: 1,3-butanediol, 1,4-hexadien, l-methoxy-2-methyl-benzen, l-octen-3-on, 1-penten, 2(5H)-furanon, 2-methyl-isoborneol, 3,3-dichlor-l-propen, 3-butyn-l-ol, 3-heptanol, 3-heptanon, 3-methyl-2-butanol, 3-methylhexan, 4-heptanol, 4-methyl-2-hexanon, caryophyllen, dimethyltrisulfid, eremophilen, Germacren D, isoleden, longifolen, methyl-2-ethylhexanoat, muurolan, terpinolen.

8. Fremgangsmåde ifølge et af kravene 1 til 7, kendetegnet ved at målmolekylet er specifikt for mere end to Aspergillus-stammer og fortrinsvis vælges fra gruppen omfattende: 1,4-pentadien, 4-heptanon, dimethyldisulfid, methoxybenzen.

9. Fremgangsmåde ifølge et af kravene 1 til 8, omfattende et trin med søgning efter områder med svampekontaminering udført før trin (a).