METHOD AND DEVICE FOR DETERMINING THE PRESENCE OF A MICRO-ORGANISM IN STOOLS WITH ACTIVATED CARBON PRETREATMENT

Abstract

The invention provides a determination method for determining the presence of a target microorganism in a patient from a sample of said patient's stools, the method being characterized in that it comprises the following operations: obtaining a sample of liquid stools of said patient or a liquid sample obtained from stools of said patient, referred to as the liquid sample; pretreating the liquid sample with activated carbon; and using immunochromatography to detect in the resulting pretreated liquid sample the possible presence of at least one antigen of the target microorganism so as to come to a conclusion about the presence or the absence of the target microorganism in said patient.

The invention also provides a device (1) for detecting an antigen of a target microorganism in the liquid sample (3) by immunochromatography, the device including a zone (20) for purification with activated carbon (21).

Claims

1. A determination method for determining the presence of a target microorganism in a patient from a sample of said patient's stools, the method being characterized in that it comprises the following operations: obtaining a sample of liquid stools of said patient or a liquid sample obtained from stools of said patient, referred to as the liquid sample; pretreating the liquid sample with activated carbon; and using immunochromatography, also known as lateral flow immunoassay, with an immunochromatography device having a sample application zone, a marking zone, and a reaction zone, to detect from the resulting pretreated liquid sample the possible presence of at least one antigen of the target microorganism so as to come to a conclusion about the presence or the absence of the target microorganism in said patient.

2. A determination method according to claim 1, characterized in that the pretreatment with activated carbon includes putting the liquid sample into contact with the activated carbon and separating the liquid sample as obtained thereby from the activated carbon.

3. A determination method according to claim 1, characterized in that it includes preparing the liquid sample of stools from said stools of said patient, and in that the pretreatment with the activated carbon is performed while preparing the liquid sample by mixing together stools, a liquid diluant, and activated carbon, followed by separating, enabling the liquid sample to be recovered without stools and without the activated carbon.

4. A determination method according to claim 1, characterized in that the pretreatment is performed by mixing the liquid sample with activated carbon followed by separating, enabling the liquid sample to be recovered without the activated carbon.

5. A determination method according to claim 1, characterized in that separating is performed by filtering.

6. A determination method according to claim 1, characterized in that the pretreatment is performed by passing the liquid sample over activated carbon positioned in a sampler device for taking the sample or in the immunochromatography device.

7. A determination method according to claim 1, characterized in that the liquid sample contains a diluant comprising a buffer, a denatured charge protein, and a detergent.

8. A determination method according to claim 1, characterized in that the microorganism is selected from viruses, bacteria, and parasites, and is preferably a rotavirus, an adenovirus, or more preferably a norovirus.

9. A determination method according to claim 1, characterized in that detection consists in detecting the interaction of at least one antigen for the microorganism of interest with at least one binding partner for binding to said at least one antigen, said binding partner preferably being an antibody or an antibody fragment.

10. A device (I, 1) for detecting at least one antigen of a target microorganism in a liquid sample (3), the device comprising: a) a support (100); and b) a porous diffusion medium (2, 200) fixed on the support (100), and enabling the liquid sample (3) to migrate, said diffusion medium (2, 200) comprising: i) an application zone (10, 300) for applying the liquid sample (3); ii) a purification zone (20, 300) including activated carbon (21, 301); iii) a marking zone (30, 500) including at least a first marked binding partner, said first binding partner being capable of binding with said at least one antigen of the microorganism that is to be detected, if present in the liquid sample, then forming a first binding partner and antigen complex; and iv) at least one reaction zone (40, 800) comprising: a display zone (41, 600) for displaying the results of the detection and comprising at least one second binding partner held stationary on the diffusion medium and suitable for binding with said first binding partner and antigen complex; a migration verification zone (42, 700) enabling proper operation of the device to be verified, which zone is situated downstream from the display zone (41, 600); and said application zone (10, 300), purification zone (20, 300), marking zone (30, 500), and reaction zone (40, 800) being in communication to enable the liquid to diffuse.

11. A device (I, 1) according to claim 10, characterized in that the first and second binding partners are antibodies or antibody fragments and/or the diffusion medium is a fiber material, in particular made of cellulose fibers or of glass fibers.

12. A detection method according to claim 1, characterized in that it uses a device (I) according to claim 10.

Description

[0072] Such devices are shown in FIGS. 1 and 2B.

[0073] FIG. 1 is a diagrammatic plan view of an example of a detector device of the invention.

[0074] FIG. 2A is a diagrammatic perspective view of a prior art detector device, and FIG. 2B is a diagrammatic perspective view of an example of a detector device in accordance with the invention.

[0075] The device 1 shown in FIG. 1 enables the liquid sample to migrate in the direction f.sub.1 and it includes a diffusion medium 2 that is fixed on a support, not shown. In known manner to the person skilled in the art, the support is water-repellant, e.g. in the form of a thin layer of plastics material. The function of the support is to stiffen, facilitate handling, and protect the diffusion medium 2. The support also serves to make the bottom face of the diffusion medium 2 waterproof and consequently to channel the flow of the sample through the diffusion medium 2.

[0076] The diffusion medium 2 has a plurality of successive zones in the direction f.sub.1, corresponding to the direction in which the liquid sample 3 that is to be deposited will migrate: the application zone 10 for applying the liquid sample 3; the purification zone 20 having activated carbon 21; the marking zone 30 having at least a first marked binding partner; the display zone 41; and the migration verification zone 42, these latter two zones constituting the reaction zone 40. Usually, at its end opposite from the application zone 10, the device 1 includes an absorption zone 50 for enhancing diffusion of the liquid sample 3. The application zone 10 and the purification zone 20 may be the same zone. The diffusion medium 2 may be constituted by a single layer of a porous matrix, or more usually, by a plurality of layers of a porous matrix, each of the layers comprising one or more zones. Any type of material that is capable of ensuring that a fluid flows and is transferred may be used as the porous matrix. The fluid may be transferred by the force of capillarity. It is possible to use a bibulous material, e.g. of the filter paper membrane type, that absorbs liquid easily and through which the liquid is transported by capillarity. In general, such a device is placed in a cassette or box (not shown) that includes a well for depositing the sample in the application zone and a window for viewing the reaction zone.

[0077] The application (and purification) zone and/or the marking zone and/or the absorption zone may in particular be constituted by porous layers fitted on, or partially overlapping on, a first layer deposited over the entire surface of the support, or more usually a first layer deposited on a portion only of the surface of the support. This first layer deposited on the support serves as an analytic membrane and it incorporates the reaction zone (display zone and migration verification zone).

[0078] Thus, in order to facilitate fabrication of the device and enhance diffusion of deposited liquid sample (in the direction f.sub.2 in FIG. 2), the various zones of the diffusion medium are constituted by a plurality of layers of a porous matrix that overlap and that are in fluid flow communication, as shown in FIG. 2B, which is described in detail when describing the examples. In particular, it is possible to use different layers of membranes or of filter paper. In particular, as shown in FIG. 2B, a layer corresponding to the purification zone 300, and also to the application zone, including activated carbon 301, may partially overlap a layer corresponding to the marking zone 500. The layer corresponding to the marking zone 500 can in turn overlap the analytic membrane 400 incorporating the display zone 600 and the migration verification zone 700. The analytic membrane 400 may extend over the entire surface of the support 100, as shown in FIG. 2B, or over a portion only of the support. Such overlaps are conventionally used in the state of the art (see in particular WO 2012/172232 and WO 2008/018073) and they make it possible in particular to ensure continuity of the flow of the liquid sample.

[0079] The difference between the device of the invention as shown in FIG. 2B and the prior art device as shown in FIG. 2A is the presence of activated carbon 301 in the zone 300.

[0080] In conventional manner, in the device of the invention, the first and second binding partners may be such as described above, and in particular antibodies or antibody fragments, and/or the diffusion medium may be a fiber material, in particular made of cellulose fibers or of glass fibers. Usually, the fiber material is made of nitrocellulose. By way of example, for the analytic membrane, it is possible to use a nitrocellulose fiber medium secured directly to a support, in particular of the polyester type.

[0081] Immunological reactions, i.e. the binding between antigens and binding partners, may be viewed by any detector means as a result of the first binding partner being marked with a marker. Thus, the first binding partner may bind marked particles that are capable of generating a detectable signal, e.g. including a marker, that may be in the form of a compound or a substance that can be detected by visual, fluorescence, or instrumental means. A non-limiting list of such markers are particles of metal or of an alloy, such as colloidal gold particles, particles of polymer, such as colored latex particles, magnetic particles, fluorescent molecules, chemiluminescent molecules, . . . . The signal generated in the result display zone and the signal generated in the migration verification zone, and that correspond to a positive result, may be of kinds that are identical or different. For example, when using colored latex, they may be of the same color or of different colors.

[0082] In a preferred implementation, the detection method of the invention uses a device as described in the present patent application.

[0083] The methods and devices of the invention for determining the presence of a microorganism in a patient are compatible with emergency situations. The symptoms of a gastroenteritis of viral origin last for only a few days, so the method and the device of the invention, when used in first intention, make it possible quickly to take the measures needed for rapidly protecting the patient and the patient's environment. The result can be interpreted within ten minutes of depositing the sample in the application zone. In care establishments, revealing norovirus by an immunochromatographic test using the method or the device of the invention leads to reinforcing hygiene measures, i.e. isolating the patient and accentuating disinfection of surfaces in order to avoid an epidemic. Furthermore, a positive result in the context of detecting a virus, makes it possible to set aside a bacterial infection, which might lead to hospitalizing elderly people, and can limit the use of antibiotics, which is pointless for a viral infection.

[0084] The examples below serve to illustrate the invention, but are of no limiting character.

Example 1: Preparing an Immunochromatographic Device for Detecting Norovirus, the Device Including a Purification Zone Based on Activated Carbon

[0085] Preparing the Device

Preparing Sample Pads

[0086] The sample pad is a strip of fiberglass having dimensions of 8 centimeters (cm)×1.7 cm that is cut from a membrane obtained from the supplier Alhstrom (Cat. No. Grade 8975, Helsinki, Finland). Prior to assembling immunochromatography strips, the sample pad is conventionally plunged into a bath of a saturation buffer.

[0087] In the control immunochromatography device (device: REF), the sample pad was plunged for 4 hours (h) in a bath having a buffer containing sugars and casein. It was then dried for 12 h to 18 h at 37° C.

[0088] In the immunochromatography device of the invention (device CARBON), the sample pad was initially subjected to the same treatment as the control immunochromatography device. Thereafter, it was plunged into a bath of a reagent based on activated carbon. The reagent based on activated carbon contained 0.65 g/L of Tris base, 6.83 g/L of Tris-HCl, 8.55 g/L of NaCl, 0.05% of Tween® 20, 1% of bovine serum albumin, and 10 g/L of activated carbon (Norit CN1, from Norit Nederland BV, Amerstoort, Netherlands). The pH was adjusted to 7.2 prior to adding the activated carbon. After 1 h, the sample pad was removed from the bath, placed on a grid, and stove-dried for 1 h at 37° C.

Preparation of the Conjugate Pad, Marking Zone

[0089] 400 nm particles of red PL-Latex Carbonyl HiDyenRed latex sold by the supplier Agilent Technologies (Cat. No. PL6104-614#) were coated by adsorption using a mixture of two immunoglobulin G (IgG) (anti-norovirus rabbit polyclonal antibodies, namely the antibodies 542 and 544 (bioMérieux). After incubating for 12 h to 18 h at ambient temperature, the particles were saturated with a casein-based buffer in order to avoid non-specific adsorptions. The particles were then spread over a fiberglass support (Cat. No. Grade 8975, Alhstrom Helsinki, Finland), which was dried overnight at 37° C.

Preparation of the Analytic Membrane, Reaction Zone

[0090] Three mouse monoclonal anti-norovirus antigen antibodies, namely clones 1H3C3, 11H12, and 2A7 (bioMérieux) were mixed together and diluted in a PBS solution. The solution as prepared in this way was spread using a BIODOT (trade name) appliance on a membrane of nitrocellulose supported by polyester reinforcement (Unisart® CN 140 backed, Sartorius, Cat. No. 1UN14ER050020), thus constituting the display zone, referred to as the test line (T). An anti-rabbit IgG polyclonal antibody was diluted in a PBS buffer. That solution was spread over the migration verification zone, situated downstream from the display zone, and referred to as the verification line (C). The membrane was then stove-dried for 12 h to 18 h at 37° C., and then conserved in a sealed aluminum pouch with a dehydrating sachet in order to preserve it from moisture.

Cassette Preparation

[0091] The strips were made by depositing the following on the nitrocellulose analytic membrane as supported in this way: a sample pad acting as a zone for applying the sample and for purifying it (fiberglass membrane acting as a filter for the sample before contact with the particles); the conjugate pad (marking zone); and an absorbent pad (an absorbent having the ability to adsorb the remainder of the sample after migrating along the various types of pad, and acting as an absorption zone). This pad was made using oil filter paper from the “automotive filter paper” range of the supplier Hangshou Xinhua Paper Industry Co. Ltd. (Hangzhou Tonglu, China). The assembly was then cut into strips having a width of 4 millimeters (mm).

[0092] The immunochromatography device of the invention (device: CARBON) is shown in FIG. 2B and differs from the device shown in FIG. 2A (device: REF) only by the fact that the first zone 300 acting both as a sample application zone and as a purification zone includes activated carbon 301. These devices enable the liquid sample to migrate in the direction f.sub.2 and they include a multilayer diffusion medium 200. The multilayer diffusion medium 200 comprises an analytic membrane 400 with incorporated support 100 that forms the first layer and it includes various successive zones in the direction f.sub.2: a first zone 300 for applying and purifying the sample and including the activated carbon 301; a marking zone 500 including at least a first marked binding partner; a display zone 600; and a migration verification zone 700, these latter two zones constituting the reaction zone 800. In certain locations, the diffusion medium is a multilayer medium. It should be observed that the layer corresponding to the application zone 300 on which the sample is deposited overlaps a layer that includes the marking zone 500. The layer including the marking zone 500 then overlaps the layer referred to as the analytic membrane 400, which includes the display zone 600 and the migration verification zone 700 for verifying flow continuity and for encouraging the liquid to diffuse by capillarity. At the end opposite from the zone 300, the device has an absorption zone 900 positioned on the layer 400 and serving to encourage diffusion of the sample.

[0093] Thereafter, these strips are individually sealed in plastics cassettes each having a sample well and a display window, ready for use.

Operating Mode of the Immunochromatography Test

[0094] Remove the cassette from its sachet and place it on a surface that is clean and flat; [0095] Deposit 75 microliters (μL) of the sample for testing in the sample well; and [0096] Start a timer in order to read the test 10 min after deposition.

[0097] The status of the sample is determined as a function of the presence or absence of a colored line of variable intensity on the nitrocellulose membrane. The intensity of the colored line varies from L1 (no red line) to L10 (an intense red line is present). By comparing it with a read card, it is possible to make an objective evaluation of the intensity of the color of the colored line.

[0098] The results are interpreted in accordance with Table 1 below:

TABLE-US-00002 TABLE 1 Reading Results Interpretation Line of intensity < L4 Negative Looked-for analyte absent Line of intensity = Positive, but Looked-for analyte L4 or L5 close to the is a priori detection limit present of the kit Line of intensity > L5 Positive Looked-for analyte is present

Detecting Norovirus by Immunochromatography

[0099] The stools are individually diluted in the VIKIA® Rota/Adeno diluant (Cat. No. 31111, bioMérieux) using 25 milligrams (mg) of stools in 1500 μL of diluant, and then vortexed for 15 s, with 75 μL being deposited in the well of the cassette. The results are given in Table 2 below.

TABLE-US-00003 TABLE 2 Stools positive for norovirus* Sample code 87 437 Device REF L3 L5 Device CARBON L4  16 *Stools of patients infected by norovirus.

[0100] The use of the immunochromatography device of the invention having a purification zone incorporating activated carbon serves to improve the detection of antigens in both of the samples selected above.

Example 2: Using a Sampling Device with a Filter During the Pretreatment of the Stool Samples, and then Detecting Norovirus by Immunochromatography

[0101] Preparing the Stool Sampling Device with a Filter

[0102] The prototype was made from the sampling tube (tube with a green stopper) in the VIKIA® Rota/Adeno kit (Cat. No. 31111, bioMérieux). The white sampling wand contained in the green stopper was removed, a Whatman GF/B (1 μm filter) having a diameter of 6 mm (Cat. No. 1821110) was installed in the bottom of the stopper, and then the end of the wand (remote from the sample) was replaced in the stopper in order to hold the filter in place. The purpose of the filter was to retain the particles of carbon and to obtain a sample suitable for placing directly in the well of the cassette.

Pretreatment of Stools and Immunochromatography

[0103] Three stool pretreatment methods were compared: [0104] Method 1: REF

[0105] The stools were individually diluted in VIKIA® Rota/Adeno diluant (Cat. No. 31111, bioMérieux) at 50 mg of stools for 3000 μL of diluant. The stools were vortexed for 15 s. 75 μL of the sample as obtained in that way were deposited in the well of the immunochromatography cassette. [0106] Method 2: CENTRIFUGING (treated with carbon+centrifuging) and Method 3: INVENTION

[0107] The reagent based on activated carbon as used in Example 1 was used again. The stools were individually diluted in the carbon reagent and 50 mg of stools per 3000 μL of diluant, and then: [0108] Method 2: they were vortexed for 15 s and then centrifuged for 5 min at 12,000 g; and [0109] Method 3: they were spread in the sampler device with the previously prepared filter.

[0110] In all of the methods, after breaking the nipple present at the end of the stopper, 75 μL of filtrate was deposited in the well of the cassette.

[0111] Table 3 below summarizes the conditions.

TABLE-US-00004 TABLE 3 Method 1 Method 2 Method 3 Stools diluted using Stools diluted with carbon reagent VIKIA ® Rota/Adeno diluant (REF) VIKIA ® 3000 μL Carbon reagent 3000 μL Rota/Adeno diluant Stools 50 mg Stools 50 mg Vortex 15 seconds Vortex 15 seconds — — — — Centrifuging 5 min Filtering Preparation 12,000 g spread in the prototype Deposit 75 μL Deposit 75 μL Deposit 75 μL

[0112] In each method, the volume of the deposit was 75 μL and reading was performed 10 min after depositing the sample. The tests were carried out using a device REF as described in Example 1, and they were read and interpreted in the same manner as in Example 1. The results are given in Table 4 below.

TABLE-US-00005 TABLE 4 Method 3 INVENTION Stools Method 1 Method 2 diluted in REF CENTRIFUING carbon Stools Stools reagent + diluted in diluted in filtering VIKIA ® carbon 1 μm Rota/Adeno reagent + filter diluant centrifuging Diameter Stopper Reference Reference 0.6 cm Control VIKIA ® L1 11 L1 sample Rota/Adeno diluant Samples E8909 GII.3 L5 L8 L7-8 positive for E9982 GII.4 L4 L9 L6 norovirus* E9918 GI.4 L4 L7 L6-7 E8624 GI L8 L9 L9 *stools of patients infected with norovirus. [0113] The use of carbon does not degrade specificity. [0114] Samples close to the detection limit (L4/L5) under the reference condition (Method 1) are very positive with the carbon treatment (Methods 2 and 3). [0115] The intensity of the read strip is greater for samples treated with carbon. [0116] For positive samples, when using carbon treatment, the test line appeared more quickly and the membrane was cleaner (the stools no longer color the membrane). [0117] The results obtained by treating the samples with a carbon-based diluant confirm the improved sensitivity. [0118] Against all expectations, the use of activated carbon, without a centrifuging step, makes it possible to obtain results that can be interpreted clinically in identical manner to the results obtained when a centrifuging step is used. In both methods, the results are positive (L≧6), whereas with the reference, the results were a priori positive. Separating by filtering instead of by centrifuging enables satisfactory sensitivity to be conserved.