Microorganism sampling method, microorganism sampling device and sampling kit comprising such a sampling device

A sampling method using a sampling device (2) comprising a container (5) comprising a body (6) consisting of a soft pouch, and a lid (30), in which at least one among the container (5) and the lid (30) is provided with a discharging member (40) suitable for, in an open state, discharging at least one part of the gases contained in the internal space (7) of the body (6) of the container (5), wherein the sampling method comprises the steps consisting in: —collecting a biological material in the body (6) of the container (5), and assembling the lid (30) on the neck (10) of the container (5), —and placing an internal space (7) of the body (6) of the container (5) under anaerobic conditions by placing the discharging member (40) in the open state, by compressing the body (6) of the container (5) and by placing the discharging member (40) in a closed state.

FIELD OF THE INVENTION

The invention relates to a microorganism sampling method, a microorganism sampling device and a sampling kit employing such a sampling device

The invention applies in particular to the sampling of human intestinal microbiota for example for testing intestinal dysbioses, such as infections ofClostridium difficile, by transportation of the intestinal microbiota.

BACKGROUND OF THE INVENTION

The intestinal microbiota is constituted by the group of all microorganisms (bacteria, yeast and fungi) to be found in the human gastro-intestinal tract (intestine, stomach and stools). The microbial diversity is currently estimated at about 103bacterial species composing the dominant intestinal microbiota of an adult individual, with an abundance of 1014bacteria, representing a bacterial metagenome of 200 000 to 800 000 genes in each individual, which is 10 to 50 times the number of genes of the human genome.

The intestines are sterile in utero and are colonized as of the first days of life to develop towards a unique individual microbiota. Each person has bacteria that are relatively close in terms of species, but the exact composition of his or her microbiota (species, proportions) is to a large extent (about ⅔ of the species) specific to the host.

Thus, the human intestinal microbiota is a very diversified ecosystem, which is complex and specific to each individual.

It is essential for the health of an individual to maintain a stable microbiota which is both capable of returning to its initial state after a change and resistant to invasion. Maintaining a wide diversity of microbiota promotes its stability.

However, certain pathologies or treatments unbalance the microbiota: for example, antibiotics as well as diseases with an inflammatory component, such as inflammatory bowel disease (IBD), can limit the diversity of the microbiota in the intestines. Antibiotics treatments (or antibiotic therapy), in particular, result in an alteration of the microbiota, which can promote the proliferation of pathogenic organisms such asClostridium difficile. Infections ofClostridium difficileare responsible for nosocomial diarrhea; this bacterium is resistant to conventional antibiotic therapy (of broad spectrum, such as vancomycin and metronidazole).

In order to reestablish the intestinal flora, and fight against infections ofClostridium difficiletype, and thereby reestablish homeostasis (i.e. symbiosis), a transplantation of faecal microbiota has been envisioned and tested. It consists in the introduction of the stools of a healthy donor subject into the digestive tract of a recipient patient, in order to re-balance the altered intestinal flora of the host. This transplantation of faecal microbiota can be allogenic (that is to say from a healthy donor individual to a patient) or autologous (that is to say from an individual to himself). The results obtained on infections ofClostridium difficiletype are encouraging, and some patients have been successfully treated (Tauxe et al, Lab Medicine, Winter 2015, volume 46, Number 1).

Prior to performing such a transplantation, the microbiota is generally sampled from the healthy donor subject through use of a sampling device of the type comprising:a container comprising a body which comprises an internal space configured to receive the biological matter, and a neck which delimits an access opening to the internal space of the body, the body of the container being constituted by a flexible bag,a cover configured to be removably and sealingly mounted on the neck of the container so as to obturate the access opening of the neck and close the internal space of the body,a transfer member internally comprising a passage between the internal space and an external environment, the transfer member having an open state in which said transfer member establishes communication with the internal space through the passage, and a closed state in which said transfer member prevents all communication with the internal space through the passage.

A known sampling device of this type is described in document WO 2013/090825. With such a sampling device, after having collected the biological matter, the container is closed with the cover before placing the biological matter in suspension by mixing the collected biological matter with a diluent fluid. Anaerobic conditions may be obtained in the internal space of the body by suction, possibly complemented by the addition of appropriate substances.

However, the known sampling device does not provide the conditions enabling a simple way of providing satisfactory preservation of the bacteria of the microbiota, and especially of the anaerobic bacteria, which are the majority components of the intestinal microbiota. Furthermore, the known sampling device does not make it possible to perform the necessary manipulations for sampling the microbiota in a safe way, the operator performing the manipulations running the risk of entering into contact with the biological matter.

The invention is directed to mitigating the problems raised above.

SUMMARY OF THE INVENTION

To that end, according to a first aspect, the invention provides a sampling method for sampling microorganisms contained in biological matter, the sampling method employing a sampling device of the aforementioned type wherein said at least one transfer member comprises an evacuation member configured for evacuating, in the open state, at least part of the gases contained in the internal space of the body of the container, the sampling method comprising the steps consisting of:collecting the biological matter in the internal space of the body of the container, and closing the internal space by mounting the cover on the neck of the container,putting the internal space of the body of the container under anaerobiosis by putting the evacuation member in the open state, by compressing the body of the container so as to evacuate at least part of the gases contained in the internal space of the body of the container by putting the evacuation member in the closed state.

Thus, the invention enables optimal conditions for the preservation of microorganisms, and in particular the microbiota, to be obtained simply and safely. In particular, the invention enables placing under anaerobiosis to be performed by simple compression applied on the body of the container to evacuate the oxygen and preserve the anaerobic bacteria that are sensitive to oxygen. The bacteria maintained in a closed system may be kept in these conditions of anaerobiosis while being isolated from external contaminants. Furthermore, this placing under anaerobiosis as well as the later placing in suspension of the biological matter that are carried out by compressing the body of the container, without having recourse to a mixer, enable the viability of the bacteria to be enhanced. Furthermore, the manipulations may be carried out with the container closed by the cover and thus without risk of direct contact with the biological matter for the operator.

the evacuation member may have opposite internal and external faces, the evacuation member being in the closed state when at rest and passing into the open state when a difference of pressure is applied between the internal and external faces, the sampling method may then provide, during the step consisting of putting the internal space of the body of the container under anaerobiosis, of automatically putting the evacuation member in the open state by compressing the body of the container then automatically putting the evacuation member in the closed state by stopping compressing the body of the container.

the cover may comprise a bearing part having overall rigidity and the evacuation member may be provided in the bearing part of the cover. The sampling method may then provide, during the step consisting of putting the internal space of the body of the container under anaerobiosis, for pressing the body of the container against the bearing part of the cover.

The sampling method may further comprise the step consisting of suspending the biological matter by introducing a diluent fluid into the internal space of the body of the container via said at least one transfer member in the open state, and by mixing the biological matter and the diluent fluid by pressing on the body of the container.

The sampling method may further comprise the step consisting of sampling the microorganisms by collecting at least some of the biological matter suspended via said at least one transfer member in the open state.

The sampling method may provide, during the step consisting of collecting the biological matter in the internal space of the body of the container, for directly collecting the faecal matter by placing the sampling device on a toilet seat.

According to a second aspect, the invention provides a sampling device for sampling microorganisms contained in biological matter, the sampling device comprising:a container comprising a body which comprises an internal space configured to receive the biological matter, and a neck which delimits an access opening to the internal space of the body, the body of the container being constituted by a flexible bag,a cover configured to be removably and sealingly mounted on the neck of the container so as to obturate the access opening of the neck and close the internal space of the body,

wherein at least one of the container and the cover is provided with at least one transfer member internally comprising a passage between the internal space and an external environment, the transfer member having an open state in which said transfer member establishes communication with the internal space through the passage, and a closed state in which said transfer member prevents all communication with the internal space through the passage,

wherein said at least one transfer member comprises an evacuation member having opposite internal and external faces, the evacuation member being in the closed state when at rest and passing into the open state when a difference of pressure is applied between the internal and external faces, so as to automatically put the evacuation member in the open state by compressing the body of the container to evacuate at least part of the gases contained in the internal space of the body of the container and place the internal space of the body of the container under anaerobiosis, and to automatically put the evacuation member in the closed state by stopping compressing the body of the container.

To avoid spreading germs and thus ensure the safety of the manipulator, the evacuation member may comprise a microporous retaining membrane disposed in the passage and configured to retain the biological matter while allowing gases to pass

Said at least one transfer member may comprise a connection port provided through one of the container and the cover and internally comprising at least part of the passage, and an obturating member movable relative to the connection port between an obturating position, in which said obturating member prevents all communication through the passage, and a freeing position in which said obturating member allows communication through the passage.

The obturating member may be mounted in the passage of the connection port, the obturating member being urged towards its obturating position and being movable towards the freeing position These provisions make it possible to make a connection simply and without boring, which is removable and fluid-tight and which, where required, enables bidirectional communication while maintaining the internal space entirely closed so long as the obturating member is not moved by a positive action towards the freeing position.

Said at least one transfer member may comprise at least one tube extending from the connection port and internally comprising part of the passage.

Said at least one transfer member may comprise at least one valve mounted on the tube, the valve being movable between a closed position, in which said valve prevents all communication through the passage, and an open position in which said valve allows the communication through the passage.

When the obturating member is mounted in the passage of the connection port, the tube may comprise a complementary connection port provided with a freeing member and which is configured to be removably connected to the connection port such that the freeing member moves the obturating member towards the freeing position.

The cover may comprise a bearing part having overall rigidity and the evacuation member may be provided in the bearing part of the cover, so as to be able to press the body of the container against the bearing part of the cover to put the internal space of the body of the container under anaerobiosis.

The sampling device may further comprise a filter attached to the container so as to define, in the internal space, an upper compartment into which the access opening opens, and a lower compartment, the filter having pores comprised between 0.1 mm and 1.5 mm.

In order to avoid obstruction of the passage by a solid body contained in the biological matter or sampling of such a solid body with the microorganisms, said at least one transfer member may be provided in the lower compartment, downstream of the filter relative to the access opening.

According to a third aspect, the invention concerns a sampling kit comprising:a sampling device as defined above, andat least one ancillary device chosen from a supply device configured to supply the internal space with fluid, and a receiving device configured to receive a fluid contained in the internal space, the ancillary device being in particular chosen from a reservoir of diluent fluid, an analysis tube, a distribution pipe and a collecting bag for microorganisms.

The sampling kit makes it possible in particular to sample and process the microorganisms into the form of an inoculum ready for use.

When the transfer member of the sampling device comprises an obturating member mounted in the passage of a connection port, the ancillary device may comprise a complementary connection port provided with a freeing member and which is configured to be removably connected to the connection port such that the freeing member moves the obturating member towards the freeing position.

DETAILED DESCRIPTION OF THE INVENTION

In the drawings, the same references designate identical or similar parts.

FIG. 1represents an embodiment of a sampling kit1comprising a sampling device2for sampling microorganisms contained in biological matter. In the embodiment represented, without being limited thereto, the sampling device2is implemented in the sampling of intestinal microbiota contained in the faecal matter of an individual.

The sampling device2comprises a container5adapted in particular to collect faecal matter.

The container5comprises a body6constituted by a flexible bag delimiting an internal space7configured to receive the biological matter and having an upper edge8. The body6is formed from a deformable material configured to form a barrier to the faecal matter contained in the internal space7and to external fluids in order to avoid any exchange between the faecal matter and the exterior. In particular, the body6may be produced from polyethylene (PE), polypropylene (PP), polyvinyl chloride (PVC), polycarbonate (PC) or ethylene-vinyl acetate (EVA).

The container5also comprises a cylindrical neck10, defined by a solid of revolution about a central axis A, to which the upper edge8of the body6is joined by any appropriate means and in particular by welding, bonding or other means. The neck10has overall rigidity and is, for example, produced from plastic material such as polyethylene (PE), polypropylene (PP), polyvinyl chloride (PVC), polycarbonate (PC) or ethylene-vinyl acetate (EVA). The neck10has an internal surface11which delimits an access opening12to the internal space7of the body6, and an external surface13provided with one or more ramps14arranged so as to form an external screw thread. As a variant, any other continuous or discrete arrangement of one or more ramps14, which may be equally spaced, could be provided on the external surface13of the neck10.

The container5also comprises a support15adapted to provide stable holding in an appropriate orientation, in particular with the access opening12upwardly oriented. In the embodiment represented, the support15is joined to the neck10and shaped so as to be able to come to bear on a rim of a toilet seat3in accordance with the application considered. The support15comprises two wings16extending radially relative to the central axis A in diametrically opposite directions from the external surface13of the neck10. As a variant, according to the application, the support could have any other appropriate shape.

A filter20is joined, for example by welding, bonding or other method, to the container5so as to define, in the internal space7, two compartments. InFIG. 1, the filter20is then joined to the body6so as to extend in the neighborhood of the access opening12of the neck10. The internal space7then has an upper compartment into which opens the access opening12and a lower compartment in the region of the bottom9. As a variant, the filter may be joined at any other appropriate location of the neck or of the body of the container to define lower and upper compartments of appropriate respective capacities. The filter has pores comprised between 0.1 mm and 1.5 mm, in particular between 0.3 mm and 0.5 mm, configured to retain insoluble solid bodies contained in the faecal matter.

The body6of the container5comprises a first transfer member forming, in the particular embodiment represented, a collecting member25enabling the microorganisms from the intestinal microbiota to be collected. The collecting member25comprises a lower connection port26formed through the body6of the container5in the neighborhood of the bottom9of the body6, and a tube27extending from the lower connection port26. The lower connection port26and the tube27thereby define a passage inside the collecting member25between the internal space7and an external environment. The collecting member25comprises a valve28mounted on the tube27and movable between:a closed position defining a closed state of the collecting member25in which it prevents any communication, and in particular any fluid flow, through the passage, andan open position defining an open state of the collecting member25in which it allows communication, and in particular fluid flow, through the passage.

As a variant, the collecting member25could comprise several tubes27and several valves28.

The tube27may be removably linked to the lower connection port26. An obturating member in the form of a check valve may be mounted in the passage of the lower connection port26and be urged towards an obturating position, in which the obturating member prevents any communication through the passage, and be movable towards a freeing position in which the obturating member allows the communication through the passage. The tube27then comprises a complementary connection port configured to cooperate with the lower connection port26. In particular, the complementary connection port is provided with a freeing member arranged to move the obturating member to the freeing position when the complementary connection port is connected to the lower connection port26. The lower connection port26and the complementary connection port may then form, one being the male member and the other the female member, a Luer Lock type connection equipped with a check valve and in which:the lower connection port26is in the obturating position preventing any communication between the internal space7and the exterior through the passage when the tube27is separated from the body6of the container5, andthe lower connection port26is in the freeing position establishing communication between the internal space7and the exterior through the passage when the tube27is connected to the body6of the container5.

Such a connection port makes it possible, by a connection without piercing, to form a two-directional communication through the passage while maintaining the internal space totally closed so long as the obturating member has not been moved towards the freeing position by the freeing member.

In another embodiment, the tube27of the collecting member25may be integrally formed with the lower connection port26, the valve28then forming the obturating member movable relative to the connection port26between an obturating position corresponding to the closed position of the valve28, and a freeing position corresponding to the open position. As a variant, instead of the valve28, the obturating member could be a plug that can be pierced or that is removably mounted on a free end of the tube27.

In order to be able to preserve the collected faecal matter, the sampling device2comprises a cover30adapted to be mounted removably and in fluid-tight manner on the neck10of the container5so as to obturate the access opening12of the neck10and to close the internal space7of the body6. The cover30has overall rigidity and is, for example, formed from a plastic material similar to that of the neck10. It comprises a transverse wall31extending perpendicularly to the central axis B and having a circular peripheral edge32with a diameter corresponding to that of the neck10of the container5. The cover30also comprises a cylindrical skirt33, defined as a solid of revolution about the central axis B, which extends from the peripheral edge32of the transverse wall31. The skirt33has an internal surface provided with projections which, according to requirement, form an internal screw thread, and which are configured to cooperate with the ramps14on the external surface13of the neck10. As a variant, any other device enabling the cover30and the neck10of the container5to be joined removably and in a fluid-tight manner could be provided, in particular a clipping device. The transverse wall31has an upper surface having two recesses34arranged on respective opposite sides of a central projection35for grasping. The recesses34are such that a lower surface of the transverse wall31at the location of the recesses34is flush with or extends beyond a free edge36of the skirt33.

In one of the recesses34, the cover30is provided with a second transfer member produced in the form of an evacuation member configured, in an open state, to evacuate at least part of the gases contained in the internal space7of the body6of the container5. In the particular embodiment represented, the evacuation member is a one-way valve40which is in a closed state at rest, when not acted upon externally, and which passes to an open state when a difference of pressure is applied between opposite internal and external faces, respectively situated at the internal and external surfaces of the cover30.

In particular, the one-way valve40comprises an upper connection port41, formed through the cover30, between its lower and upper surfaces, and a tube42extending from the upper connection port41. The upper connection port41and the tube42thereby define a passage inside the one-way valve40between the internal space7and the external environment. In a central bead43, the tube42of the one-way valve40comprises a microporous retaining membrane disposed in the passage and which is configured to retain the biological matter while enabling gases to pass. The microporous retaining membrane may in particular be of the type commercialized by POREX® FILTRATION under the reference XS-49110made of polypropylene with pores comprised between 125 μm and 175 μm. As a variant, the microporous retaining membrane could be produced in any other appropriate way and in particular of a sintered material having pores comprised between 100 μm and 350 μm, in particular between 200 μm and 300 μm, for example 250 μm.

An obturating member in the form of a check valve may be mounted in the passage of the one-way valve40and be urged towards an obturating position, preventing any communication through the passage, and be movable towards a freeing position allowing communication through the passage.

In the embodiment shown, the tube42of the one-way valve40is integrally formed with the upper connection port41. The obturating member may be provided in the tube42, in particular in the neighborhood of a free end.

In another embodiment, the tube42of the one-way valve40may be removably connected to the upper connection port41. In addition to or instead of the check valve provided in the tube42, a new obturating member in the form of a check valve may be provided in the upper connection port41and be urged towards an obturating position, preventing any communication through the passage, and be movable towards a freeing position allowing communication through the passage. In the this other embodiment, the tube42comprises a complementary connection port adapted to cooperate with the upper connection port41. In particular, the complementary connection port is provided with a freeing member arranged to move the check valve of the upper connection port41to the freeing position when the complementary connection port is connected to the upper connection port41. The upper connection port41is in the obturating position when the tube42is separated from the cover30and in the freeing position when the tube42is connected to the cover30.

As a variant, the evacuation member could be formed in any appropriate way other than a one-way valve. In particular, the evacuation member could comprise a passage able to be obturated by an obturating member in the form of a plug removably placed in the passage, for example by being removably mounted on the free end of the tube42. When the plug is removed from the passage, the evacuation member is in the open state, in which communication between the internal space7of the container5and the exterior is established through the passage. When the plug is placed in the passage, it obturates the passage and the evacuation member is in a closed state, in which any communication between the internal space7of the container5and the exterior through the passage is prevented.

In the other recess34, the cover30comprises third and fourth transfer members which respectively form, in the particular embodiment shown, a supply member45and a sampling member48. The supply member45and sampling member48are each adapted to establish communication between the internal space and the exterior in the open state, and to close the communication between the internal space and the exterior in the closed state.

The supply members45and sampling member48respectively comprise additional upper connection ports46,49for ancillary devices of the sampling kit.

Each of the additional upper connection ports46,49is formed through the cover30, between the lower and upper surfaces, to constitute one of the members, for example female, of a Luer Lock type connection. As was described above, each of the additional upper connection ports46,49of the supply member45and sampling member48may be provided with an obturating member produced in the form of a check valve47, a plug or other member.

The obturating member of the supply member45is, for example, produced in the form of a check valve47mounted in the passage and configured to cooperate with a freeing member of a complementary connection port provided on the ancillary device and constituting the other member, for example male, of the Luer Lock type connection. The check valve47urged towards an obturating position preventing any communication through the passage places the additional upper connection port46of the supply member45in a closed state when the ancillary device is separated from the cover30. The obturating member47moved to a freeing position permitting communication through the passage places the additional upper connection port46of the supply member45in an open state when the ancillary device is connected to the cover30.

The obturating member of the sampling member48is, for example, produced in the form of a plug, not shown, removably mounted on the additional upper connection port49. The sampling member48is in an open state promoting communication through the passage when the plug is separated from the additional upper connection port49, and a closed state preventing any communication through the passage when the plug is on the additional upper connection port49.

The ancillary devices may in particular comprise:one or more reservoirs of diluent fluid, and in particular a supply bag, configured to be connected to the additional upper connection port46of the supply member45, the reservoir comprising, where required, a pipe equipped with a complementary connection port with a freeing member.one or more analysis tubes configured to be connected to the additional upper connection port49of the sampling member48, each analysis tube extending between an open end provided, where required, with a complementary connection port with a freeing member, and a closed end so as to be able to extract faecal matter for analysis purposes, andone or more bags for collecting microorganisms configured to be connected to the lower connection port26of the collecting member25via a distribution duct comprising one or more pipes equipped with a complementary connection port with a freeing member and, where required, with one or more valves.

As a variant, the sampling kit1could comprise any other type of ancillary device chosen from a supply device configured to supply the internal space7with fluid, and a receiving device configured to receive a fluid contained in the internal space7.

The invention has been described with a sampling device comprising first, second, third and fourth transfer members respectively constituting a collecting member, an evacuation member in the form of a one-way valve, a supply member and a sampling member. As a variant, it would be possible to provide any other arrangement, any other embodiment and any other functionality with one or more transfer members internally comprising a passage and of which at least one forms an evacuation member configured, in the open state, to evacuate at least part of the gases contained in the internal space of the body of the container. In particular, the sampling device2could comprise only the first25and second40transfer members described earlier or for instance only one of them. At least one transfer member is provided, preferably in the lower compartment, downstream of the filter relative to the access opening, in order to avoid obstruction of the connection port by a solid body contained in the biological matter or a sample of such a solid body with the microorganisms.

A method of sampling microbiota contained in biological matter implementing the sampling system will now be described in relation withFIGS. 2 to 4.

InFIG. 2, faecal matter is collected by placing the container5of the sampling device2on the toilet seat3with the wings resting on the rim of the toilet seat3. As a variant, the faecal matter could be deposited in any other appropriate way in the internal space7of the container5. During this collecting step, the tube27may be separated from the container5.

InFIG. 3, once the faecal matter has been received on the filter20in the internal space7of the body6, the internal space7is closed by screwing the cover30onto the neck10of the container5. The collecting member25, the one-way valve40and the two sampling members45,48are in the closed state. An operator may then proceed with placing the internal space7of the body6of the container5under anaerobiosis by compressing the body6of the container5thereby automatically passing the one-way valve40into the open state. In particular, the transverse wall31of the cover30at the location of the recesses34forms a bearing part against which the body6of the container5can be pressed when the cover30is mounted on the neck10of the container5. In doing this, part of the gases contained in the internal space7of the body6of the container5, and in particular the oxygen, is evacuated through the one-way valve40provided in the bearing part of the cover30. The compression of the body6may be improved by a grip or rest on a gripping member, such as a rigid plate or a handle, arranged on a base9at the remote opposite to the upper edge8. Once the compression of the body6of the container5is stopped, the one-way valve40automatically passes back to the closed state to maintain the anaerobic conditions in the internal space7of the body6.

According to the application considered, the placing under anaerobiosis may be completed by an injection of inert gas into the internal space7of the body6of the container5via one of the transfer members25,40,45,48.

After a possible waiting time under determined conditions and possible visual inspection, the faecal matter is placed in suspension by connecting the reservoir of diluent fluid, such as a bag of diluent fluid or a bottle of diluent fluid, to the additional upper connection port46of the supply member45on the cover30of the sampling device2. The additional upper connection port46passes from the open state such that the diluent fluid is introduced into the internal space7of the body6of the container5. The biological matter and the diluent fluid are then mixed by pressing on the body6of the container5. The homogeneity of the suspension is improved by its passage through the filter20.

Prior to the placing in suspension of the faecal matter, complementary analyses may be performed on a sample taken via an analysis tube connected to the additional upper connection port49of the sampling member48on the cover30of the sampling device2. As a variant, the sampling of such a sample may be performed simultaneously or subsequently to the placing in suspension.

To process the microbiota into the form of an inoculum ready for use, the tube27or a distribution duct may be connected to the lower connection port26of the collecting member25to take off the microbiota contained in the faecal matter to one or more collecting bags respectively connected to one or more pipes of the distribution duct.

The microbiota may then undergo any appropriate operation for its preservation, possible transport and its transplantation.