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WO2008126111A1 - Test rapide de chimiosensibilité d'helicobacter pylori - Google Patents

Test rapide de chimiosensibilité d'helicobacter pylori Download PDF

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Publication number
WO2008126111A1
WO2008126111A1 PCT/IT2007/000270 IT2007000270W WO2008126111A1 WO 2008126111 A1 WO2008126111 A1 WO 2008126111A1 IT 2007000270 W IT2007000270 W IT 2007000270W WO 2008126111 A1 WO2008126111 A1 WO 2008126111A1
Authority
WO
WIPO (PCT)
Prior art keywords
culture medium
sample
test
helicobacter pylori
antibiotic
Prior art date
Application number
PCT/IT2007/000270
Other languages
English (en)
Inventor
Federico Perna
Dino Vaira
Original Assignee
Alma Mater Studiorum-Università Di Bologna
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Alma Mater Studiorum-Università Di Bologna filed Critical Alma Mater Studiorum-Università Di Bologna
Priority to PCT/IT2007/000270 priority Critical patent/WO2008126111A1/fr
Publication of WO2008126111A1 publication Critical patent/WO2008126111A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/56922—Campylobacter
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/18—Testing for antimicrobial activity of a material

Definitions

  • the present invention relates to a method for the determination of the chemosusceptibility of the bacterium Helicobacter pylori.
  • the invention refers to a simple, rapid and effective method for the identification of the antibiotic susceptibility of Helicobacter pylori.
  • Helicobacter pylori 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.
  • biochemical assays urease, catalase, oxidase, etc.
  • the colonies are picked up using a loop and dispersed in a saline solution at a defined concentration.
  • 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.
  • 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) .
  • the methods just described despite being the methods of reference, are very laborious and hence poorly used for practical purposes.
  • 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.
  • 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.
  • the method for determining the antibiotic susceptibility of Helicobacter pylori comprises of the steps of :
  • 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;
  • the culture medium selected as a result of its improved efficacy, is prepared at the time ("in house") from the individual constituent components .
  • 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 :
  • antibiotics such as vancomycin, trimethoprim, cefsulodin, amphotericin B, polymyxine B.
  • 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.
  • 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;
  • 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 +4 0 C, 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 37 0 C in the presence of one or more antibiotics which need to be tested for chemosusceptibility) .
  • the quantitative or semiquantitative analysis stage is preferably carried out using a direct ELISA or EIA test specific for Helicobacter pylori.
  • 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) .
  • the individual components for an ELISA or EIA 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) .
  • the individual components for an ELISA or EIA test can be any
  • 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.
  • the advantages of the present invention are significant .
  • 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.
  • the presence of Helicobacter pylori is determined with confidence, thus becoming also a diagnostic method, and not useful for performing a chemosusceptibility only.
  • 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.
  • 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 (XXT 7 TMB etc.) in place of an ELISA or EIA type test.
  • 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 (XXT 7 TMB etc.) in place of an ELISA or EIA type test.
  • the method for determining the antibiotic chemosusceptibility of Helicobacter pylori comprises the steps of:
  • 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;
  • 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.
  • culture medium is identical to that previously described with particular reference to the specifications of BBLB medium.
  • a semiquantitative immunochromatographic test it might be possible to use a semiquantitative immunochromatographic test. This is to make a method even faster and simpler to execute.
  • 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.
  • the method for determining the antibiotic chemosusceptibility of Helicobacter pylori comprises the steps of:
  • pancreatic digest of casein OXOID, lp0042
  • the medium will be sterilised in an autoclave and once having reached the temperature of approx. 50 0 C the following components can be added:
  • 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;
  • 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.
  • 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.
  • a heating plate to 37 0 C can be used for the tubes (perhaps also with spaces at room temperature or at 4 0 C for the TO tube) to avoid the need for a 37 0 C incubator.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Health & Medical Sciences (AREA)
  • Hematology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Food Science & Technology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Toxicology (AREA)
  • Biophysics (AREA)
  • Virology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Medicinal Chemistry (AREA)
  • Cell Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne un procédé pour déterminer la chimiosensibilité de la bactérie Helicobacter pylori. L'invention se rapporte notamment à un procédé consistant à identifier la chimiosensibilité antibiotique d'Helicobacter pylori de manière simple, rapide et efficace en utilisant un milieu de culture particulier avec un test de détection quantitatif/semi-quantitatif. Le procédé susmentionné permet de réduire le laps de temps nécessaire qui est de 6 à 11 jours requis pour obtenir un test de chimiosensibilité en utilisant les procédés connus, à seulement 20-24 heures.
PCT/IT2007/000270 2007-04-12 2007-04-12 Test rapide de chimiosensibilité d'helicobacter pylori WO2008126111A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/IT2007/000270 WO2008126111A1 (fr) 2007-04-12 2007-04-12 Test rapide de chimiosensibilité d'helicobacter pylori

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/IT2007/000270 WO2008126111A1 (fr) 2007-04-12 2007-04-12 Test rapide de chimiosensibilité d'helicobacter pylori

Publications (1)

Publication Number Publication Date
WO2008126111A1 true WO2008126111A1 (fr) 2008-10-23

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102559565A (zh) * 2012-03-07 2012-07-11 重庆理工大学 选择性培养幽门螺旋杆菌的混合抑菌剂及其制备方法
CN104833804A (zh) * 2015-04-30 2015-08-12 必欧瀚生物技术（合肥）有限公司 一种幽门螺杆菌IgG抗体ELISA半定量检测试剂盒及其应用
CN105820975A (zh) * 2016-03-25 2016-08-03 美利泰格诊断试剂(嘉兴)有限公司 一种口腔幽门螺杆菌培养方法
JP2019071853A (ja) * 2017-10-18 2019-05-16 北海道公立大学法人 札幌医科大学 抗菌剤のスクリーニング方法

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999032656A1 (fr) * 1997-12-23 1999-07-01 Consortia Laboratories Evaluation colorimetrique de la sensibilite d'helicobacter pylori a des substances antimicrobiennes

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999032656A1 (fr) * 1997-12-23 1999-07-01 Consortia Laboratories Evaluation colorimetrique de la sensibilite d'helicobacter pylori a des substances antimicrobiennes

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
M. KIST ET AL: "Pathogenesis, diagnostics and treatment of Helicobacter pylori infection", BUNDESGESUNDHEITSBLATT, GESUNDHEITSFORSCHUNG, GESUNDHEITSSCHUTZ JUN 2005, vol. 48, no. 6, June 2005 (2005-06-01), FRG, pages 669 - 678, XP002464489, ISSN: 1436-9990 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102559565A (zh) * 2012-03-07 2012-07-11 重庆理工大学 选择性培养幽门螺旋杆菌的混合抑菌剂及其制备方法
CN102559565B (zh) * 2012-03-07 2014-07-02 重庆理工大学 选择性培养幽门螺旋杆菌的混合抑菌剂及其制备方法
CN104833804A (zh) * 2015-04-30 2015-08-12 必欧瀚生物技术（合肥）有限公司 一种幽门螺杆菌IgG抗体ELISA半定量检测试剂盒及其应用
CN105820975A (zh) * 2016-03-25 2016-08-03 美利泰格诊断试剂(嘉兴)有限公司 一种口腔幽门螺杆菌培养方法
JP2019071853A (ja) * 2017-10-18 2019-05-16 北海道公立大学法人 札幌医科大学 抗菌剤のスクリーニング方法

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