WO2008126111A1

WO2008126111A1 - A chemosusceptibility rapid test for helicobacter pylori

Info

Publication number: WO2008126111A1
Application number: PCT/IT2007/000270
Authority: WO
Inventors: Federico Perna; Dino Vaira
Original assignee: Alma Mater Studiorum-Università Di Bologna
Priority date: 2007-04-12
Filing date: 2007-04-12
Publication date: 2008-10-23

Abstract

The present invention relates to a method for the determination of the chemosusceptibility of the bacterium Helicobacter pylori. In particular, the invention refers to a method consisting of identifying the antibiotic chemosusceptibility of Helicobacter pylori in a simple, rapid and effective manner by using a particular culture medium along with a quantitative/semiquantitative detection test . The above mentioned method allows reducing the time required from the 6-11 days required to obtain a chemosusceptibility test using the known methods, to 20-24 hours only.

Description

DESCRIPTION

^WA CHEMOSUSCEPTIBILITY RAPID TEST FOR HELICOBACTER

PYLORI"

FIELD OF THE INVENTION [0001] . The present invention relates to a method for the determination of the chemosusceptibility of the bacterium Helicobacter pylori. In particular, the invention refers to a simple, rapid and effective method for the identification of the antibiotic susceptibility of Helicobacter pylori.

BACKGROUND OF THE INVENTION

[0002] . One of the methods currently used to identify the antibiotic susceptibility of the bacterium Helicobacter pylori consists of performing a chemosusceptibility test predominantly based on culture agar-based media with different types of supplements . [0003] . In particular, the method starts with a biopsy sample taken from the stomach and streaked onto agar plates. The micro-organism is allowed to grow (isolation) by means of incubation at 37°C in microaerophily for 3-7 days (using jars with suitable kits for creating necessary environment) . Once grown, the isolated bacterium is identified by eye or by means of biochemical assays (urease, catalase, oxidase, etc.) and/or gram staining. Once isolated and identified as Helicobacter pylori, the colonies are picked up using a loop and dispersed in a saline solution at a defined concentration. At this point, using a swab soaked in the saline solution in which the Helicobacter pylori was spreaded, the bacterium is once more seeded on agar (spreading the swab in 3 directions) where an antibiotic- soaked disk (disk diffusion) , or a strip with an increasing antibiotic concentration gradient (E-test) is placed. Again, in this case, incubation is carried out at 37°C in a controlled environment. After 3-4 days, the chemosusceptibility test is "read" in an entirely conventional manner.

[0004] . Another method consists of dissolving the antibiotic directly in the agar (agar dilution) to assess whether, at a defined concentration, the bacterium grows (resistant) or is inhibited (susceptible) . [0005] . The methods just described, despite being the methods of reference, are very laborious and hence poorly used for practical purposes. [0006] . Indeed, performing a standard chemosusceptibility test requires at least 3-7 days for the first isolation and a further 3-4 days from the reseeding of the isolated bacteria with the addition of antibiotic. Therefore, a total of 6-11 days overall are necessary. SUMMARY OP THE INVENTION

[0007] . Hence, the technical problem resolved by the present invention is that of providing a faster method for determining the antibiotic susceptibility of the bacterium Helicobacter pylori.

[0008] . This problem is resolved by a method for determining the chemosusceptibility of Helicobacter pylori to antibiotics, as stated in the main claim appended hereto . [0009] . Further characteristics and the advantages of the method will become more evident from the following description of an embodiment of the present invention, given by way of non-limiting example. DETAILED DESCRIPTION OF THE INVENTION [0010] . The idea underlying the present invention is that of devising a detection system that allows the quantitative assessment of the bacteria grown on a suitable culture medium that is much faster compared to the analysis times according to the known art. [0011] . Based on the aforementioned surmise, it has been proposed to exploit a quantitative or semiquantitative detection method that is known but has never been applied to determinate a chemosusceptibility test . [0012] . Following numerous experiments, it has been found that the method lending itself best to this type of analysis is a test capable of detecting the presence of Helicobacter pylori antigens, such as ELISA or EIA tests. [0013] . Furthermore, the samples to be assayed, grown as usual on agar, must be picked up from a solid medium using a sterile loop and resuspended by means of a laborious procedure which slows-down and complicates the entire procedure.

[0014] . Hence, a method for optimally preparing the samples for the aforementioned analysis has been studied. For this reason, various different culture media have been studied, and a particular liquid culture medium has been selected. The choice of a specific liquid culture medium in place of conventional culture media has shown itself to be surprisingly advantageous, since it has been shown to allow improved percentage growth without interfering with the activity of the antibiotic under test. Furthermore, execution of the entire chemosusceptibility procedure is simplified and speeded up. [0015] . Hence, in accordance with the present invention, the method for determining the antibiotic susceptibility of Helicobacter pylori comprises of the steps of :

- preparing a liquid culture medium comprising growth factors, such as those present in inactivated FCS (Foetal Calf Serum) and/or HS (horse serum) and/or FBS (foetal bovine serum) and/or horse blood and/or sheep blood and/or nutrients which can replace blood and serum, selective antibiotics, vitamins, mineral salts, a source of carbohydrate, protein, amino acids, minerals, coenzymes, and optionally, bisulphites;

- adding a biological sample to above mentioned liquid culture medium along with at least one antibiotic under test, with optional prior breaking-up of the above sample;

- leaving the biological sample in the liquid culture medium for a sufficient period of time and under optimal conditions for achieving the desired growth;

- removing an aliquot of the sample and subjecting it to quantitative-semiquantitative analysis by means of tests capable of identifying the presence of Helicobacter pylori antigens. [0016] . The culture medium, selected as a result of its improved efficacy, is prepared at the time ("in house") from the individual constituent components . In particular, a liquid culture medium which may contain the aforementioned components is known by the initials BBLB (Brocella Broth Less Bisulphite) , and is specifically composed of :

- inactivated FCS;

- pancreatic digest of casein;

- peptidic digest of animal tissue; - dextrose;

- yeast extract ;

- sodium chloride;

- vitamin B₁₂, L-glutamine, adenine, guanine hydrochloride, p-aminobenzoic acid, NAD, thiamine pyrophosphate, ferric nitrate, thiamine hydrochloride, L-cysteine hydrochloride, L-cysteine

- selective antibiotics such as vancomycin, trimethoprim, cefsulodin, amphotericin B, polymyxine B. [0017] . The antibiotic or the antibiotics to be tested are added to the medium once the sample to be tested has been added. The antibiotics may be selected from those normally used in standard therapy against Helicobacter pylori, such as clarithromycin or metronidazole, or may be novel antibiotics or any substance being assayed for inhibitory and/or bactericidal activity.

[0018] . Preferably, 500 mL of the liquid culture medium is constituted by: - 5 g of pancreatic digest of casein; - 5 g of peptidic digest of animal tissue; - 0.5 g of dextrose;

- 1 g of yeast extract;

- 2.5 g of sodium chloride;

- 5 mg of vancomycin, 2.5 mg of trimethoprim, 2.5 mg of cefsulodin, 2.5 mg of amphotericin B (purchased individually or as a bottle of Helicobacter pylori selective supplement commercially available by OXOID (trade name Helicobacter Selective Supplement, code SRO147E) _/ - 10000 IU of Polymyxine B (purchased from any supplier or OXOID -trade name Bacillus Cereus Selective Supplement, code SR99E- in particular, adding 1/5 of this supplement containing 50000 IU/tube) _/

- 3.3 mL of ISOVITALEX^® (Becton-Dickinson code 211875) or VITOX^® (OXOID code SR0090A) ;

- 25 mL of inactive FCS;

- 475 mL of distilled water.

[0019] . Moreover, in particular, the test comprises a stage of dividing up the sample, once mixed with the liquid culture medium, into N equal parts, where one part is frozen or stored at +4⁰C, one part assigned to growth by incubation in microaerobiosis or anaerobiosis or normal atmosphere (at 37°C without the antibiotics to be tested and one or more parts are assigned to growth in a controlled atmosphere at 37⁰C in the presence of one or more antibiotics which need to be tested for chemosusceptibility) .

[0020] . The quantitative or semiquantitative analysis stage is preferably carried out using a direct ELISA or EIA test specific for Helicobacter pylori. In particular, the test can be any commercially available ELISA or EIA test such as for example the Premier Platinum HpSA Plus ELISA kit (code 601396) commercially available by Meridian, or the Ridascreen Femtolab H. pylori kit (code 2301) commercially available by Femtolab Connex or also by DIA.pro Diagnostic (code Ag. ce) . Alternatively, the individual components for an ELISA or EIA test

(solutions, antibodies, marker molecules) can be purchased and made "in house" using antibodies such as those commercially available by Signet or other commercially available antibodies.

[0021] . It should be noticed that ELISA or EIA kits and methods are standard in almost all analysis and research laboratories, and so will not be described in any further detail here. As a non-limiting example, we cite the test described in Perna, Vaira et al., Helicobacter 2004; 9 (5) : 436-442.

[0022] . The advantages of the present invention are significant . [0023] . In particular, the method for determining the antibiotic susceptibility of Helicobacter pylori above described, surprisingly makes it possible to reduce the times required for the culture and the chemosusceptibility from 6-11 days to just 24 hours. Furthermore, by means of the antibody used for the specific test, the presence of Helicobacter pylori is determined with confidence, thus becoming also a diagnostic method, and not useful for performing a chemosusceptibility only. [0024] . It should be highlighted that this method also allows rapid evaluation of the susceptibility to novel compounds by simply dissolving the novel substance in the medium, or allows monitoring the synergic effect of several substances by analysing the sample with the substances individually, and the sample where several substances have been mixed together.

[0025] . In addition, an advantageous synergic effect has been demonstrated by the combination of an ELISA or EIA type test with the particular liquid culture medium specified previously. Indeed, the method allows the isolation, identification and accurate assessment of chemosusceptibility of Helicobacter pylori with a single test. [0026] . Among the advantages of this test, it should also be stressed that the culture of Helicobacter pylori, being long and laborious, requires times which can cause contaminants, such as moulds or other bacteria to grow, thus preventing isolation. On the other hand, the method in accordance with the present invention, being very rapid and based on a specific antibody, excludes a priori interference from other micro-organisms potentially capable of also growing on the medium used despite being selective .

[0027] . In accordance with a variant embodiment of the invention, the above-described method may be accomplished by using another detection method such as for example an assay for urease (CP-test or rapid urease test) or other markers such as catalase, oxidase or commercially available cell viability markers (XXT₇ TMB etc.) in place of an ELISA or EIA type test. This allows to decrease costs and reduce the times typical of ELISA tests, while maintaining a colourimetric type test .

[0028] . Consequently, the method for determining the antibiotic chemosusceptibility of Helicobacter pylori comprises the steps of:

- preparing a liquid culture medium, with or without bisulphites, comprising inactivated FCS (Foetal

Calf Serum) or other substances such as those described previously (HS, FBS, serum-free media) , selective antibiotics, vitamins, mineral salts, a source of carbohydrates, protein, amino acids, minerals, coenzymes;

- adding a biological sample to said mentioned liquid culture medium along with at least one antibiotic to be tested, with optional prior breaking-up of said sample;

- leaving the biological sample in the liquid culture medium for a sufficient period of time and under conditions for achieving the desired growth; - removing an aliquot of the sample and assigning it to analysis by means of methods suitable for identifying the count and/or viability of

HeIicobacter pylori .

[0029] . Preferably, as mentioned above, the methods suitable for identifying the count or the viability of Helicobacter pylori are selected from assays for the activity of urease (CP-test or rapid urease test) , catalase, oxidase or commercially available cell viability markers such as XXT (XXT Serva code 38450) , TMB (TTC Solution Fluka code 17779-10X10ML-F) tests etc.

Furthermore, the culture medium is identical to that previously described with particular reference to the specifications of BBLB medium.

[0030] . According to one additional variant embodiment of the invention, it might be possible to use a semiquantitative immunochromatographic test. This is to make a method even faster and simpler to execute. For example, it might be possible to devise a quantitative or semiquantitative (strip type test) immunochromatographic test which stains with one or more bands depending on the bacterial load found; this test, by comparing a sample at to (time zero) and after 20-24 hours with or without the antibiotic to be tested might, like the ELISA test, allow evaluating whether the bacteria has grown or not and consequently whether or not it is resistant.

[0031] . It should be observed that the method for determining the antibiotic chemosusceptibility of Helicobacter pylori as described up to now, might be advantageously applied to samples of faeces or saliva (to make the test non-invasive) or gastric fluid instead of a biopsy fragment, by appropriately modifying the growth medium.

[0032] . In the latter case, the method for determining the antibiotic chemosusceptibility of Helicobacter pylori comprises the steps of:

- adding a biological sample to a liquid culture medium comprising at least one antibiotic to be tested, with optional prior breaking-up of the sample;

- leaving the biological sample in the liquid culture medium for a sufficient period of time and under conditions for achieving the desired growth;

- removing an aliquot of the sample and assigning it to semiquantitative analysis by means of ELISA or EIA type tests. [0033] . It should be remembered that the method of the invention might be applied (by altering the medium and/or growth conditions and/or temperature and/or specific antibodies) for the determination of the chemosensitivity of other bacteria or mycetes, normally studied by conventional type tests.

[0034] . An example embodiment of the invention, given purely by means of non-limiting example is reported hereinafter . EXAMPLE Preparation of the biological sample in BBLB liquid culture medium with 5% FCS

A biopsy sample, taken during and endoscopic examination of the antral area of the stomach, is first broken up using standard scalpels. Afterwards, the fragments are placed in 800 μL of BBLB with 5% FCS, prepared by mixing in 475 mL of distilled water to give a total of 500 mL:

- 5 g of pancreatic digest of casein (OXOID, lp0042) ;

- 5 g of peptidic digest of animal tissue (OXOID, IpO037) ; - 0.5 g of dextrose (OXOID, lpOO71) ; - 1 g of yeast extract (OXOID, lp0021) ;

- 2.5 g of sodium chloride (OXOID, lp005) ;

At this point, the medium will be sterilised in an autoclave and once having reached the temperature of approx. 50⁰C the following components can be added:

- one jar of Helicobacter pylori selective supplement commercially available by OXOID under the name of Helicobacter Selective Supplement, code SR0147E containing 5 mg of vancomycin, 2.5 mg of trimethoprim, 2.5 of cefsulodin, 2.5 of amphotericin B;

- 1/5 of Bacillus cereus selective supplement containing 50000 IU/jar commercially available by OXOID under the name of Bacillus Cereus Selective Supplement, code SR99E;

- 3.3 mL of ISOVITALEX^® (Becton-Dickinson code 211875) or VITOX^® (OXOID code SRO09OA) ;

- 25 mL of inactivated FCS (GIBCO BRL- Life technologies 10270-106, Batch 40g2810k, inactivated for 30 minutes at 56°C) .

[0035] . At this stage, after having mixed the liquid medium containing the fragments, it is split into 4 equal volume aliquots (i.e. 200 μl) in 4 test tubes. The first is stored at 4⁰C (TO) , the second incubated at 37°C in microaerobic environment (T-20) , to the third one is added clarithromycin (T-CH) at a final concentration of 2 μg/mL, while to the fourth one is added metronidazole (T-MZ) at a final concentration of 8 μg/mL. The latter two tubes containing antibiotics are also incubated in microaerobic environment at 37°C along with the T-20 tube. [0036] . After 20-24 hours, the tube labelled TO and the other three tubes incubated at 37°C will be used to perform into four liquid media ELISA tests using, in this case, the Premier Platinum HpSA ELISA kit (code 601348, not currently on the market) commercially available by Meridian.

[0037] . Finally, by means of colourimetric reading, an optical density (O.D.) is obtained, as with all ELISA tests. At this point, the ratio has been established between the various optical densities, in particular: [(0.D. of sample T-CH or T-MZ) - (O.D. of sample TO)] /

[(0.D. of sample T20) - (O.D. of sample TO)] . From analysis of the data, with respect to the "gold standard", namely Agar Dilution, a "cut off" value has been established to allow to decide whether the bacterium is susceptible or resistant to one antibiotic or the other. The cut off value calculated takes into consideration the Premier Platinum HpSA ELISA test (code 601348) , currently not on the market, but which can be replaced by the Premier Platinum HpSA Plus kit (code 601396) commercially available by Meridian, to devise an appropriate cut off, or in the case of using an ELISA kit from another company or "in house" kit, it is necessary to recalibrate the cut off.

[0038] . For the experiment, a 37⁰C incubator has been used, along with the OXOID microaerophily kit (code CN0025A) , and jars for sealing the incubation tubes. Identification of the cut off and the sensitivity of the method of the invention with respect to the GOLD STANDARD method [0039]. In order to verify that the new method is working and to assess the sensitivity and specificity, a study has been performed using 105 biopsy samples. These have been assessed for chemosusceptibility to clarithromycin and metronidazole using the gold standard AGAR DILUTION method and the new method. In relation to clarithromycin, 75 sensitive cultures and 30 resistant cultures have been identified. Taking the new method into consideration, all 75 have been sensitive using the cut off specified below (sensitivity of the method - 100%) , while 28 out of 30 have been identified as resistant, and two have been falsely sensitive (method specificity - 93.3%). In relation to metronidazole, 68 sensitive cultures and 37 resistant cultures have been identified. Taking the new method into consideration, 63 have been sensitive and 5 falsely resistant (method sensitivity - 92.6%), while 32 out of 37 have been identified as resistant, and 5 have been falsely sensitive (method specificity - 86.5%).

[0040] . In this experiment, the above Meridian kit has been used with a cut-offs of >55 and >27 (as the above- described ratio of optical densities) to identify those resistant to clarithromycin and metronidazole respectively. [0041] . From the above description, it is obvious that the previously described problems associated with the methods of the known art have been resolved, and at the same time, a number of advantages have been provided, above all in terms of the rate of response of the method and the simplicity of execution. [0042] . In any case, those skilled in the art can make further modifications to the present method, while remaining within the scope of protection of the invention as defined in the following claims. [0043] . For example, with the present method for determining the susceptibility to the Helicobacter pylori antibodies, the use of stoppers allowing the formation of microaerobic environment in the test tubes, in place of the use of jars and kits for microaerophily, or conducting the method directly in a normal atmosphere, can be considered. At this point, a heating plate to 37⁰C can be used for the tubes (perhaps also with spaces at room temperature or at 4⁰C for the TO tube) to avoid the need for a 37⁰C incubator.

Claims

1. A method for determining the antibiotic susceptibility of Helicobacter pylori wherein a biological sample is grown in a liquid culture medium as defined, said grown biological sample being subsequently subjected to a test capable of identifying the presence of Helicobacter pylori antigens, such as ELISA, comprising the stages of:

- preparing a liquid culture medium, with or without bisulphites, comprising inactivated FCS (Foetal Calf Serum) and/or HS (horse serum) and/or FBS

(foetal bovine serum) and/or horse blood and/or sheep blood and/or nutrients which can replace blood and serum, selective antibiotics, vitamins, mineral salts, a source of carbohydrate, protein, amino acids, minerals, coenzymes; adding a biological sample to said liquid culture medium along with at least one antibiotic to be tested, with optional prior breaking-up of said sample; - leaving the biological sample in said liquid culture medium for a sufficient period of time and under optimal conditions for achieving the desired growth; removing an aliquot of said sample and assigning it to quantitative-semiquantitative analysis by means of tests capable of identifying the presence of Helicobacter pylori antigens.

2. The method according to claim 1, wherein said mentioned determination of antibiotic susceptibility of Helicobacter pylori is performed within 20-24 hours.

3. The method according to claims 1 or 2 , wherein said medium consists of :

- inactivated FCS;

- pancreatic digest of casein; - peptidic digest of animal tissue;

- dextrose;

- yeast extract;

- sodium chloride;

- vitamin Bi₂, L-glutamine, adenine, guanine hydrochloride, p-aminobenzoic acid, NAD, thiamine pyrophosphate, ferric nitrate, thiamine hydrochloride, L-cysteine hydrochloride, L-cysteine;

- selective antibiotics such as vancomycin, trimethoprim, cefsulodin, amphotericin B, polymyxine B.

4. The method according to claims 1, 2 or 3, wherein 500 mL of said medium consists of:

- 5 g of pancreatic digest of casein;

- 5 g of peptidic digest of animal tissue; - 0.5 g of dextrose; - 1 g of yeast extract;

- 2.5 g of sodium chloride;

- one jar of Helicobacter pylori selective supplement commercially available by OXOID under the name of Helicobacter Selective Supplement, code SR0147E containing 5mg of vancomycin, 2.5 mg of trimethoprim, 2.5 mg of cefsulodin, 2.5 mg of amphotericin B;

- 1/5 of Bacillus cereus selective supplement (containing 50000 IU/tube) equivalent to 10000 IU, commercially available by OXOID under the name of Bacillus Cereus Selective Supplement, code SR99E_/

- 3.3 niL of ISOVITALEX (Becton-Dickinson code 211875) or VITOX (OXOID code SRO09OA) ;

- 25 rtiL of inactive FCS; - 475 mL of distilled water.

5. The method according to any of the claims 1 to 4 , further comprising a stage of dividing up the sample, once mixed with the liquid culture medium, into N equal parts, where one part is frozen or stored at +4⁰C, one part subjected to growth in microaerobic at 37⁰C without the antibiotics to be tested, and one or more parts are subjected to growth in microaerobic at 37°C in the presence of one or more antibiotics to be tested.

6. The method according to any of the claims 1 to 5, wherein said quantitative or semiquantitative analytical stage is achieved by means of an ELISA or EIA type test.

7. The method according to claim 6, wherein said test is a test carried out using the Premier Platinum HpSA Plus ELISA kit (code 601396) commercially available by Meridian or the Ridascreen Femtolab H. pylori kit (code 2301) commercially avalaible by Femtolab Connex or also by DIA.pro Diagnostic (code Ag. ce) .

8. The method according to any of the claims 1 to 6, wherein the antibody used for the ELISA test is Rabbit Polyclonal Pylori commercially avalaible by Signet.

9. A method for determining the antibiotic chemosusceptibility of Helicobacter pylori comprises the steps of:

- preparing a liquid culture medium according to any of the claims 1, 3 or 4;

- mixing a biological sample in said mentioned liquid culture medium along with at least one antibiotic to be tested, with optional prior breaking-up of said sample; - leaving the biological sample in said liquid culture medium for a sufficient period of time and under optimal conditions for achieving the desired growth; removing an aliquot of said sample and assigned it to analysis by means of methods suitable for identifying the count and viability of Helicobacter pylori.

10. The method according to claim 9, wherein said methods suitable for identifying the viability of Helicobacter pylori comprise the evaluation of the activity of the enzyme urease (CP-test or rapid urease test) , catalase, oxidase or cell viability markets such as the XXT or TMB tests.

11. A method for determining the antibiotic chemosusceptibility of bacteria or mycetes comprising the steps of:

- mixing a biological sample in said mentioned liquid culture medium comprising at least one antibiotic to be tested, with optional prior breaking-up of said sample; leaving the biological sample in the liquid culture medium for a sufficient period of time and under optimal conditions for achieving the desired growth; - removing an aliquot of said sample and assigning it to semiquantitative analysis by means of an ELISA test.

12. The method according to claim 11, wherein said bacterium is Helicobacter pylori.

13. The method according to any of the claims 1 to 11, wherein the biological sample to be subjected to antibiotic chemosusceptibility analysis is a biopsy fragment, a sample of faeces, saliva, gastric fluid or urine .

14. A method for determining the antibiotic chemosusceptibility of bacteria or mycetes comprising the steps of:

- preparing a liquid culture medium according to any of the claims 1, 3 or 4; - adding a biological sample said liquid culture medium along with at least one antibiotic to be tested, with optional prior breaking-up of said sample;

- leaving the biological sample in said liquid culture medium for a sufficient period of time and under optimal conditions for achieving the desired growth; bringing said culture medium, comprising said grown sample, into contact with a support for a quantitative or semiquantitative immunochromatographic test which stains with one or more bands, depending on the bacterial load;

- comparing said support, after contact, with an analogous support brought into contact with a sample at to (time zero) and after 20-24 hours without test antibiotic in order to assess whether the bacterium has grown or not, and consequently, whether or not it is resistant.

15. The method according to claim 13 , wherein said bacterium is Helicobacter pylori.