WO2021220302A1 - Procédé pour déterminer la sensibilité du bacille de koch à un médicament sur un disque en papier - Google Patents

Procédé pour déterminer la sensibilité du bacille de koch à un médicament sur un disque en papier Download PDF

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Publication number
WO2021220302A1
WO2021220302A1 PCT/IN2021/050414 IN2021050414W WO2021220302A1 WO 2021220302 A1 WO2021220302 A1 WO 2021220302A1 IN 2021050414 W IN2021050414 W IN 2021050414W WO 2021220302 A1 WO2021220302 A1 WO 2021220302A1
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WIPO (PCT)
Prior art keywords
drug
determining
tuberculosis
paper
disc
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PCT/IN2021/050414
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English (en)
Inventor
Suman CHAKRABORTY
Srikanth Prasad Tripathy
V. N. Azgerdusthackeer
Shantimoy KAR
B. Mahizhaveni
S. Govindarajan
Original Assignee
Indian Council Of Medical Research
Indian Institute of Technology Kharagpur
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Application filed by Indian Council Of Medical Research, Indian Institute of Technology Kharagpur filed Critical Indian Council Of Medical Research
Publication of WO2021220302A1 publication Critical patent/WO2021220302A1/fr
Priority to ZA2022/11453A priority Critical patent/ZA202211453B/en

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    • 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
    • C12Q1/66—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving luciferase
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/35—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Mycobacteriaceae (F)

Definitions

  • the present invention provides a method for determining drug susceptibility of bacteria. More specifically, the present invention provides a paper disc based method for determining the drug susceptibility of M. tuberculosis. Particularly, the present invention provides a method of determining drug resistance characteristics of M. tuberculosis towards anti- microbial drugs.
  • Tuberculosis remains as an important infectious disease and public health concern worldwide. According to the latest World Health Organization (WHO) report, tuberculosis (TB) causes 10 million cases and 1.5 million deaths annually and it is estimated that 3 million cases go undiagnosed each year. Not surprisingly, multi-drug-resistant tuberculosis (MDR-TB) is a significant problem, and India now has the most number of cases of multidrug-resistant tuberculosis (MDR)-TB in the world, contributing one-fourth of the global burden. MDR-TB is caused by strains of Mycobacterium tuberculosis that are resistant to at least rifampicin and isoniazid, two key drugs in the treatment of the disease.
  • WHO World Health Organization
  • Veasna et al., 2019 describes advances in programmable microfluidics i.e. paper based continuous flow microfluidics (p-CFM) devices and paper based digital microfluidic (p-DMF) device for biomarker detection.
  • p-CFM paper based continuous flow microfluidics
  • p-DMF digital microfluidic
  • US patent 6,300,061 B1 relates to the production and use of luciferase reporter mycobacteriophages to diagnose tuberculosis.
  • the document also describes a method for screening for drugs which inhibit the growth of mycobacterial lysogens.
  • WO1994025572A1 relates to mycobacterial species-specific reporter mycobacteriophages.
  • the document describes the use of mycobacteriophages to rapidly diagnose mycobacterial infection and to assess drug susceptibilities of mycobacterial strains in clinical samples.
  • a paper disc based method for determining drug susceptibility of M. tuberculosis comprising preparing a paper disc and incubating with a sample; treating the disc of step (i) with an anti-microbial drug; adding a reporter bacteriophage to the drug treated disc and incubating at 37°C for a period of 180 minutes; and screening by addition of a substrate and measuring relative light units.
  • the present invention provides a method of determining the drug susceptibility of M. tuberculosis, wherein the paper disc is prepared from Whatmann paper (grade I).
  • the present invention provides a method for determining the drug susceptibility of M. tuberculosis, wherein the sample is mycobacterial isolate.
  • the present disclosure provides a method for determining the drug susceptibility of M. tuberculosis, wherein the antimicrobial drugs are Rifampicin or Isoniazid.
  • the present disclosure provides a method of determining the drug susceptibility, wherein the paper discs are incubated with a reporter phage.
  • the present invention provides a method of determining the drug susceptibility, wherein the substrate is D-luciferin.
  • a diagnostic kit for determining the drug resistant bacteria comprising of a paper disc treated with antimicrobial drug; a sample; luciferase reporter phage; and a substrate.
  • a method for determining the drug susceptibility of M. tuberculosis wherein the method is capable of determining the drug susceptibility in shortest period of time i.e. about 3 hours.
  • Figure 1 illustrates schematic representation of the process for paper disc based method for determining the drug susceptibility for M. tuberculosis.
  • the paper discs were incubated with the culture isolate, reporter phage and the anti-microbial drugs and thereafter D-luciferin was added. Depending on the bacterial activities in presence of drugs, the luminescence on the paper discs turns on which essentially determines the susceptible characteristics against the specific drug. DETAILED DESCRIPTION OF THE INVENTION
  • the present invention provides a paper disc based method for determining drug susceptibility of M. tuberculosis.
  • the present invention provides a paper-based microfluidic platform for detecting drug resistance characteristics of M. tuberculosis.
  • the discs are employed to investigate the drug resistance characteristics towards anti-microbial drugs rifampicin and isoniazid (. Mycobacterium tuberculosis resistant to both these drugs and is referred as multidmg resistant tuberculosis- MDR TB).
  • the present invention provides a rapid, cost-effective and less-time consuming method for determining drug resistance characteristics of M. tuberculosis towards anti-microbial drugs.
  • the process of the present invention comprises cutting the paper discs for 5mm diameter in standard laboratory grade Whatmann filter papers (grade 1). Three sterile discs are kept in a partition bi plate using a sterile syringe. The discs are labeled as Cl, C2 and C3. Cl and C2 labeled discs are used as controls and are incubated with 30 pi Middle brooke 7H9 broth containing oleic acid albumin dextrose catalase growth enrichment and 8m1 of mycobacterial culture suspension. The mycobacterial culture suspension comprises the colonies of M.
  • tuberculosis isolates scrapped from a 3 week old growth from Lowenstein Jensen slant and mixed with 7H9 broth in a bijou bottle containing glass beads of 10 to 12 numbers of 3 mm diameter. The solution is vortexed for 60 seconds and allowed to settle. Further, the supernatant is aliquoted, and the turbidity was matched with a 1.0 McFarland standard culture.
  • C3 disc is labeled with the corresponding drug Rifampicin and Isoniazid.
  • the test was performed in duplicates. In one set, C3 is treated with Rifampicin (30 m ⁇ of drug containing 7H9 broth at lpg/rnl) and in other set, C3 is treated for Isoniazid (30 m ⁇ of isoniazid containing 7H9 broth at 0.1 pg/ml concentration). Subsequently, 8m1 of mycobacterial culture suspension is added to the drug treated discs.
  • the paper discs Cl, C2 and C3 are treated with 8m1 of luciferase reporter phage construct phAETRC202 and 4 m ⁇ of 0.1 M Calcium chloride solution (CaCk ) and incubated for a period of 180 minutes at temperature of 37°C.
  • a substrate D-Luciferin was added, and the discs are transferred on the luminometer (Bethhold Technologies, Lumat, LB 9507) tube for measuring the output signals (in relative light units -RLU). The relative light units were measured using a luminometer device (Monolight 2010).
  • a paper disc based method for determining drug susceptibility of M. tuberculosis comprising:
  • step (ii) treating the disc of step (i) with an anti-microbial drug
  • the present disclosure provides a method of determining the drug susceptibility of M. tuberculosis, wherein the paper disc is prepared from Whatmann paper (grade I).
  • the process of the present disclosure uses paper discs having porous structure (i.e. cellulose fabric-based network of the paper) that provides high surface area to increase the interaction between the reaction systems; thus, suits as a better diagnostic platform.
  • porous structure i.e. cellulose fabric-based network of the paper
  • the intrinsically present capillary force of the paper matrix enhances the required reaction time and thus, reduces the overall assay time.
  • the constant evaporation of fluids from the matrix and the random porous structures in combination results in continuous redistribution of fluids (i.e. reagents/chemicals) in microscopic scales.
  • the heterogeneous distribution of reagents at local level triggers the bacterium to find the equilibrium stage, and thereby keeping it more active as compared to any conventional culture-based systems.
  • the present invention provides a method for determining the drug susceptibility of M. tuberculosis, wherein the sample is mycobacterial isolate.
  • the present disclosure provides a method for determining the drug susceptibility of M. tuberculosis, wherein the antimicrobial drugs are Rifampicin or Isoniazid.
  • the present disclosure provides a method of determining the drug susceptibility, wherein the paper discs are incubated with a reporter phage.
  • the phage to be used for the purpose of the present invention is luciferase reporter phage (LRP).
  • the phage is selected from the group consisting of phAETRC16, phAETRC21, phAETRC201 and phAETRC202.
  • the luciferase reporter phage construct is phAETRC202.
  • the reporter phage of the present disclosure is species- specific luciferase reporter bacteriophage, which are genetically engineered phages and can detect the presence of live bacilli.
  • the luciferase reporter phage infects the mycobacteria and produces luciferase enzyme, upon addition of the substrate D-luciferin.
  • the relative light units are measured using a luminometer device.
  • the assessment of drug susceptibilities with the reporter bacteriophages of the present disclosure is accurate because the reporter bacteriophages only allow for the detection of metabolically active mycobacterial organisms, the presence of which metabolic activity indicates that a drug has not killed the mycobacteria and that the mycobacteria is resistant to the drug.
  • the present invention provides a method of determining the drug susceptibility, wherein the substrate is D-luciferin.
  • the present disclosure provides a diagnostic kit for determining the drug resistant bacteria, the kit comprising of:
  • a method for determining the drug susceptibility of M. tuberculosis wherein the method is capable of determining the drug susceptibility in shortest period of time i.e. about 3 hours.
  • Colonies of M. tuberculosis isolates were scrapped from a 3 week old growth from Lowenstein Jensen slant and mixed with 7H9 broth in a bijou bottle containing glass beads of 10 to 12 numbers of 3 mm diameter. The solution was vortexed for 60 seconds and was allowed to settle. The supernatant was aliquoted and the turbidity was matched with 1.0 McFarland standard.
  • the salt solution can also be prepared by using Difco dehydrated powder by weighing 4.7 g of dehydrated base into a 2 liter flask and adding 900 ml distilled water. Further, by adding 0.5 ml of Glycerol and then mixing well. Subsequently, the solution was distributed in 95 ml amount and autoclaved at 15 lbs for 15 minutes. Further, to each 95 ml salt solution, 5 ml of sterile ADC (bovine albumin-dextrose- catalase) solution was added aseptically and mixed well. The solution was distributed in 5- 10 ml amount in sterile universal containers and was incubated overnight at 37°C to check the sterility and then stored in cold (4°C).
  • sterile ADC bovine albumin-dextrose- catalase
  • a stock solution of rifampicin was prepared at a concentration of lOmg/ml. Initially, the Rifampicin was dissolved in dimethyl formamide and the volume was made up with distilled water at a final concentration of 1 pg/ml.
  • a stock solution of Isoniazid was prepared at a concentration of lOmg/ml in distilled water. Further, the isoniazid was dissolved and sterilized with a filter and then, the volume was made up with distilled water at a final concentration of 0.1 pg/ml.
  • sterile paper discs were cut in 5mm diameter in standard laboratory Whatmann filter papers (grade 1). The sterile discs were kept in a partition bi plate using a sterile syringe. The discs were labeled as Cl and C2 were used as controls and are meant for drug free bacterial suspensions (in duplicates) and the third disc C3 was labeled with the corresponding drug i.e. RMP for Rifampicin and INH for Isoniazid, meant for drug containing the bacterial suspension.
  • RMP for Rifampicin
  • INH Isoniazid
  • the drug free discs (controls) Cl and C2 were incubated with 30 pi of Middle brooke 7H9 broth containing oleic acid albumin dextrose catalase growth enrichment (7H9) and 8m1 of mycobacterial culture suspension.
  • C3 labeled disc was treated with 30 m ⁇ of drug Rifampicin (lpg/ml) or Isoniazid (O.lpg/ml) containing 7H9 broth and 8m1 of mycobacterial culture suspension.
  • Example 3 Sensitivity assay for detecting drug susceptibility
  • the three paper discs (Cl, C2 and C3) were then incubated with the luciferase reporter phage construct phAETRC202 and 4m1 of 0.1 M Calcium chloride solution (CaCh) followed by an incubation at 37°C for a period of 180 minutes.
  • the phage was propagated and reconstituted in the Mycobacteriophage (MP) Buffer (1M Tris, 5M NaCl, 1M MgC12 and 1M CaCh).
  • the discs were then transferred to a luminometer cuvette individually and 50 pi of D-Luciferin was added [substrate containing 0.33 mM D-luciferin (R&D Systems, Minneapolis, MN, USA) in 0.05 M sodium citrate buffer at pH 4.5].
  • the relative light units were measured using a luminometer device (Monolight 2010) with 10 seconds integration time.
  • RLU Relative Light Units
  • Example 4 Drug resistance characteristics of M. tuberculosis towards anti-microbial drugs
  • the M. tuberculosis isolates were used for determining the drug resistance characteristics of M. tuberculosis towards anti-microbial drugs.
  • Drug 1 RIFAMPICIN: Time: 180 minutes
  • tuberculosis isolates were taken for Rifampicin (RMP) drug resistance detection.
  • the tests were performed using the using the gold standard method (LJ based minimal inhibitory concentration method) and it was found that 40 isolates were Rifampicin (RMP) susceptible and 14 isolates were RMP resistant. Further, the isolates were examined by the disc based method, and were able to detect 39 of the 40 drug susceptible isolates as susceptible and 13 out of 14 drug resistant isolates as resistant correctly in duration of 180 minutes.
  • Drug 2 ISONIAZID; Time: 180 minutes
  • Isoniazid (INH) detection 47 M. tuberculosis isolates were used. Among them, 26 isolates was Isoniazid (INH) susceptible and 21 isolates were Isoniazid (INH) resistant, using the conventional gold standard method (LJ based minimal inhibitory concentration method). Further, the isolates were examined by the Disc based method, which was able to detect 26 of the 26 drug susceptible isolates as susceptible and 19 out of 21 drug resistant isolates as resistant correctly in duration of 180 minutes.
  • results demonstrate that the paper disc based method of the present invention provides results in shortest duration of time. Further, the method is more sensitive and accurate.

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Abstract

La présente invention se rapporte à un procédé pour déterminer la sensibilité de bactéries à un médicament. Plus particulièrement, la présente invention concerne un procédé pour déterminer la sensibilité du bacille de Koch à un médicament sur un disque en papier, consistant à préparer un disque en papier et à l'incuber avec un échantillon ; à traiter le disque par un médicament antimicrobien ; à ajouter un bactériophage au disque traité par médicament et à l'incuber à 37 °C pendant une durée de 180 minutes ; et à cribler par addition d'un substrat et à mesurer les unités de lumière relative. Ainsi, la présente invention fournit un procédé économique, rapide et sensible pour évaluer la résistance de la tuberculose aux médicaments.
PCT/IN2021/050414 2020-04-29 2021-04-28 Procédé pour déterminer la sensibilité du bacille de koch à un médicament sur un disque en papier WO2021220302A1 (fr)

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Application Number Priority Date Filing Date Title
ZA2022/11453A ZA202211453B (en) 2020-04-29 2022-10-19 A paper disc based method for determining the drug susceptibility of mycobacterium tuberculosis

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IN202011018419 2020-04-29
IN202011018419 2020-04-29

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994025572A1 (fr) * 1993-04-29 1994-11-10 Albert Einstein College Of Medicine Of Yeshiva University, A Division Of Yeshiva University Mycobacteriophages rapporteurs specifiques d'especes mycobacteriennes
US20080241819A1 (en) * 2006-10-31 2008-10-02 Microphage (Tm) Incorporated Method and apparatus for enhanced bacteriophage-based diagnostic assays by selective inhibition of potential cross-reactive organisms

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994025572A1 (fr) * 1993-04-29 1994-11-10 Albert Einstein College Of Medicine Of Yeshiva University, A Division Of Yeshiva University Mycobacteriophages rapporteurs specifiques d'especes mycobacteriennes
US20080241819A1 (en) * 2006-10-31 2008-10-02 Microphage (Tm) Incorporated Method and apparatus for enhanced bacteriophage-based diagnostic assays by selective inhibition of potential cross-reactive organisms

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CAMPBELL, JOSHUA M ET AL.: "Microfluidic and paper-based devices for disease detection and diagnostic research", INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, vol. 19, no. 9, 2018, pages 2731, XP055871793 *
SOUM, VEASNA ET AL.: "Programmable paper-based microfluidic devices for biomarker detections", MICROMACHINES, vol. 10, no. 8, 2019, pages 516, XP055871796 *
VAN KLINGEREN, BERT ET AL.: "Drug susceptibility testing of Mycobacterium tuberculosis complex by use of a high-throughput, reproducible, absolute concentration method", JOURNAL OF CLINICAL MICROBIOLOGY, vol. 45, no. 8, 2007, pages 2662 - 2668, XP055064543, DOI: 10.1128/JCM.00244-07 *

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