FORM 2
THE PATENT ACT 1970
(39 of 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See section 10 and rulel3)
1. TITLE OF THE INVENTION:
DEVICE AND METHOD FOR DETERMINING ANTIMICROBIAL SENSITIVITY
2. APPLICANT (S)
(a) NAME: WOCKHARDT LTD.
(b) NATIONALITY: INDIAN
(c) ADDRESS: Wockhardt Research Centre, D-4, MIDC, Chikalthana,
Aurangabad, 431210
3. PREAMBLE TO THE DESCRIPTION
The present invention relates to a test device and a method for determination of minimum inhibitory concentration of an antimicrobial agent. The invention also relates to producing predefined concentration gradients of chemically and biologically active ingredients on a porous carrier.
The following specification particularly describes the invention and the manner in which it is to be performed.
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FIELD OF THE INVENTION
The present invention relates to a test device and a method for determination of minimum inhibitory concentration of an antimicrobial agent. The invention also relates to producing predefined concentration gradients of chemically and biologically active ingredients on a porous carrier.
BACKGROUND OF THE INVENTION
The determination of the minimum inhibitory concentration (MIC) of an antibiotic is an essential laboratory test to determine the sensitivity / resistance of a microorganism, to the test antimicrobial agent. The MIC refers to the minimum concentration of an antibiotic necessary to prevent the growth of a microorganism. For the treatment of a disease caused by a microorganism to a human or an animal, the type and dose of an antimicrobial agent is often decided based upon this type of test. Getting such results rapid and accurate is critical to both patient and cost-effective treatment. Some of the methods available for the determination of MICs are complicated, time consuming, expensive and tedious.
One of the methods comprises a series of two-fold dilutions of the active substance in a fluid or a solid medium. The method is known as a tube dilution method and involves placing an equal amount of a target microorganism in plurality of wells referred to as "tubes" disposed in a platter, and adding different concentrations of an antibiotic to each tube. The lowest concentration of antibiotic in which the target microorganism will not grow determines the MIC for that particular microorganism. This method has the disadvantage that it is time consuming and expensive.
Another antimicrobial susceptibility testing method is based on Kirby-Bauer plate method (Baur, A.W., Kirby, W.M.M., Sherds, J.C. and Turck, M., "Antibiotic susceptibility testing by standardized single disc method", Am. J. Clin. Pathol. 44, 493-496, 1966) which involves the diffusion of the active substance from a defined diffusion centre to an agar layer. The concentration gradients of different substances depend on the
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particular diffusion properties of the individual substances. The method does not use continuous concentration gradient. However, this method provides information related to resistance and sensitivity of micro-organism only qualitatively and does not provide MIC values.
In paper disc diffusion sensitivity method, the sensitivity of the microorganism is determined by use of antibiotic impregnated paper disc of a recommended concentration per disc. In this method, the extent of zone of inhibition surrounding the disc determines the sensitivity or resistance of that microorganism when values compared with those that are recommended by an authority. This method has limitation in a sense that we do not get absolute MIC values and the method is not recommended by CLSI for some of the antibacterial agents such as colistin.
U.S. Patent No. 4,778,758 discloses a sealable container to store strips of non-porous nature. The strips have R, I and S regions on the strip meaning if one gets values in R region then it shows that the microorganism is resistant and if it is in S region, then the organism is sensitive. This is not appropriate as sensitivity breakpoints differ from one type of microorganism to another for the same antimicrobial agent and hence one cannot make zones on the strip, common for testing sensitivity of all types of microorganisms. The '758 patent discloses some of the disadvantages associated with the porous antibiotic discs. It discloses that the porous material is unable to diffuse antibiotics uniformly due to variation in porosity of the paper and differences in the physicochemical properties of different antibacterial agents.
U.S. Patent No. 5,028,529 discloses a method and device for the qualitative and quantitative detection of antimicrobial activity. The device material comprises of a non-porous plastic material strip. The non-porous rectangular strip is used to determine MIC of an unknown microorganism with an increasing concentration of antimicrobial agent having maximum and minimum concentrations at extreme ends. These strips are commercially available as Etest®. The supporting medium is of a non-porous nature on which antimicrobial agent is applied with concentration gradient on one side of the strip
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and MIC values are calibrated on the other side of the strip. The strip has to be placed on an agar medium with the calibrated side facing upward and antimicrobial agent applied side facing downwards, on the growth supporting medium.
After incubation, two clear zones of growth inhibition on either side of the strip meet at a calibrated value, this value represent the MIC value of the test antimicrobial.
However, the Etest strip has a technical problem in reading the results sometimes. Due to the calibrated values printed only on one side of the Etest strip, it is essential to place the strip on growth supporting medium with calibrated side of the test strip facing upwards. In such a situation, on incubation of the plates in warm incubators for growth to take place, small amount of water from the aqueous growth medium condenses on the inner side of the lid when the plates are removed from the incubators. This condensed water in the form of droplets obstructs the view to the printed values on the strip. Therefore, to read the values one has to open the lid of the plates to view the finely calibrated values. The opening of the lid, without use of the safety cabinet has an inherent danger of exposing the pathogenic organism to the atmosphere. Moreover, for certain antimicrobial for example macrolides and clindamycin "dip effect" is observed with the Etest strips. Also, for large molecules like glycopeptides which bind to different matrices and diffuse very slowly in agar, despite the efficient release of the gradient from the strip into the agar, molecules remain in the vicinity of the strip. Etest strip results for Vancomycin and Teicoplanin generally results in the characteristically slim inhibition ellipses. In such cases the correct end point is to be interpreted in a very complex manner and it requires a lot of background learning and education for reading and interpretation of results. Hence, a person who is experienced and trained is required to interpret the results correctly and accurately. Furthermore, the strip is difficult to manufacture and the non-porous synthetic carrier of antimicrobial agent is not eco-friendly. These strips are expensive and hence cannot be used liberally for MIC determination in various healthcare centers and hospitals.
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There is an absolute need to have a test device for determining MIC values of an antimicrobial agent for a target microorganism, which is not only cost effective but also superior in terms of accuracy and ease of interpreting the results.
The present invention describes porous test strips with continuous gradient of the antimicrobial agent in a predetermined concentration pattern having maximum concentration at one end of the strip and minimum concentration at the other end of the strip for the determination of the MIC values.
DETAILED DESCRIPTION OF THE INVENTION
As mentioned above there are various methods reported for the determination of MIC values. In order to get reproducible results and also to have a control over possible variation in the procedures and thereby influencing MIC values, regulatory authorities recommend standard protocols that are acceptable worldwide. In the US, Clinical and Laboratory Standards Institute (CLSI), formerly National Clinical Laboratory Standards (NCCLS) recommends protocols and MIC breakpoints for various antimicrobial agents currently employed in clinics. The CLSI (CLSI, 2006, "Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically", 7th Edition, Approved Standard M7-A7, Vol. 26, No. 2) promotes the development and use of voluntary consensus standards and guidelines within the healthcare community.
It is recognized worldwide for the application of its unique consensus process in the development of standards and guidelines for patient testing and related healthcare issues in a cost effective way. The other equivalent authorities are Gut Foundation of Australia (GFA) and European Committee on Antibiotic Susceptibility Testing (EUCAST).
According to CLSI, for a particular antimicrobial agent, the MIC value against a reference organism provided by American Type Culture Collection (ATCC) should be within the MIC range as recommended by CLSI. Therefore, the MIC determination has to be performed for an unknown strain along with a control reference strains in the same
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test system. If the MIC values for the ATCC reference organism falls within the range recommended by CLSI, then under same conditions, the MIC value against the unknown strain becomes acceptable. Under clinical situation, the determination of MIC is critical to choose the most appropriate treatment option. Any error in the determination of MIC may result in improper identification of a resistant organism as a sensitive one or a sensitive as a resistant one. Hence, obtaining the correct MIC values within the recommended range of reference strains is critical and serves as an internal standard for validation of method and results.
The present invention discloses a test device for the determination of MIC values of antimicrobial agents comprising a porous carrier.
It is another object of the present invention to provide a test device, comprising a porous carrier, containing on the surface thereof at least one chemically or biologically active substance for example an antimicrobial agent, wherein substantially all of the active substances are on either side of the carrier in a continuous concentration gradient manner, so that when carrier is applied to the surface of a antimicrobial growth supporting medium, a concentration pattern is transferred to the surface of the same medium fully or partially which on further processing gives MIC values. The test device has calibrated values printed on the test device.
Another object of the present invention is to provide a method for the determination of MIC of an antimicrobial agent comprising the steps of:
a) providing a test device comprising porous test strip in a rectangular, oval or oblong shape;
b) the antimicrobial agent is applied to the porous test strip in a continuous concentration gradient from highest concentration at one end to lowest concentration at the other end;
c) providing a microorganism growth supporting medium and inoculating the medium with target microorganisms;
d) placing the porous test strip on the microorganism growth supporting medium with any side of the strip in contact with the medium;
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e) incubating the growth medium for a time period sufficient for the target
microorganism to grow;
f) determining the value corresponding on the calibrated scale where the zone of inhibition from highest concentration from either side of the test strip meet at a particular point on the strip is the MIC value of the antimicrobial agent for that target microorganism;
g) also providing a control experiment with CLSI recommended ATCC microorganism for which the value of MIC obtained from the above steps is in the CLSI recommended range.
Further advantage of the test strips of present invention is that for the macromolecules such as macrolides, clindamycin or large molecules like glycopeptides, easy to read end points are achieved due to absence of dip effect and slim inhibition ellipses.
Another embodiment of the present invention is the use of the test strips from both sides. The porous nature of the calibrated carrier allows one to apply the test strip keeping printed side facing downwards to the transparent agar media in a transparent Petri-dish. This enables to interpret results without opening the lid of a petri-dish as one can read results from the transparent bottom side of the Petri dish. However, for a non porous strip material where the calibration is done on one side, one needs to open the lid to read the results as condensed water on the inner side of the lid would obstruct the view to the reading scale. This is a dangerous situation as pathogenic organisms get exposed to open atmosphere.
The examples of porous carrier used for the present invention are either paper such as chromatography paper or rayon, nylon and other cellulose derivatives. The porous carrier may be optionally treated with suitable additive in solutions such as glucose, fructose, xylose, mannose, sodium chloride, potassium chloride, sodium citrate, water soluble starch, cyclodextrins, hydroxypropylmethyl cellulose (HPMC), Tween 80, bile salts, glycerol etc. which may help in blocking porous channels which are left open during the
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fine manufacturing process of carrier material, without hindering the antimicrobial diffusibility process.
The porous carrier also referred as test strips are rectangular, oblong or oval in shape. The test strip is graded with MIC values with the highest values at one end and lowest values at the other.
The test strips of the present invention have an advantage is that the test strips are cost effective and have the ability to provide accurate MIC values due to a continuous concentration gradient of the antimicrobial agent on the test strips.
The antimicrobial agent is applied to the specific surface of the rectangular strip with a continuous concentration gradient along the longitudinal length. The micro-delivery of the antimicrobial solution can be done manually or with the aid of automated delivery system on the specific surface of the test strip with a concentration gradient decreasing from a maximum at one end of the carrier to a minimum at the other end. The antimicrobial agent can be applied by manual or automated system for micro delivery of specific volumes of antimicrobial solutions having defined decreasing concentrations onto the surface of the carrier, thereby resulting in a continuous concentration gradient along the specific test strip area.
The antimicrobial agent is applied to the surface of the test strip by dissolving the antimicrobial in an inert solvent vehicle such as methanol, ethanol, Tween 80, water soluble polymer such as HPMC, glycerol, water or a mixture thereof which help in uniformly occupying the porous channels which are left open during the fine manufacturing process of carrier material, without hindering the diffusibility process.
The term "antimicrobial" refers to antibacterial such as aminoglycosides, cephalosporins, fluoroquinolones, macrolides, glycopeptides, oxazolidinones, polymyxins, polypeptides and other chemotherapeutics such as sulfonamides, antimycotics, for example 5-fluorocytosine, amphotericin, antifungals, antivirals agents such as adenine arabinoside
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(Ara-A), trifluorothymidine, anti-tuberculer drugs, such as isoniazide, etambutol, prizinamide and cycloserine, and disinfectants, antiseptics and preservatives such as chlorohexidine, ethanol and benzalkonium chloride so on.
The term "microorganism" refers to bacteria, such as aerobic, anaerobic, fastidious and atypical bacteria. Bacteria includes Enterobacteriaceae, staphylococci, streptococci, Hemophillus, Neisseria, Bacteroides, and Clostridia, Mycobacteria, Actinomycetes, Mycoplasma, Nocardia, virus, and fungi such as moulds, yeasts and Candida.
The term "growth supporting medium" comprises agar, gelatin as a solidifying agent and further containing growth promoting components such as peptone, yeast extract, soyabean extract, glucose or any carbohydrate source, salts, with or without buffering agents, blood or other animal organ extracts as the component as per the requirement for specific test microorganism as well as growth media recommended by eg. CLSI.
The type of growth supporting medium depending on the type of microorganism will be known to one skilled in the art.
Example-1
A typical experiment details for the determination of MIC using the test strip of the invention:
A test strip is placed with either side having printed values facing upwards or downwards on a solid growth supporting medium inoculated with target microorganism so that the surface of the strip is placed with the test organism so as to remains firmly in contact with the growth medium. Plates containing the medium alongwith the test strips are then incubated at an optimum growth inducing conditions depending on type of microorganism. The incubation time will depend upon the nature of microorganism. After incubation, the zone of inhibition emerging from highest concentration from either side of the test strip and meeting together at a particular point on the strip is observed and the value corresponding to that point is the MIC of the antimicrobial agent against that particular test microorganism, provided, in the parallel control experiment using the test
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strip of the invention, the MIC values for CLSI recommended ATCC microorganisms for that particular antimicrobial agent fall within the CLSI recommended MIC range.
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Claims:
1. A test device for making a determination of minimum inhibitory concentration
comprising a porous carrier containing on the surface thereof at least one chemically or
biologically active substance, wherein substantially all of the active substances are on
either side of the carrier in a concentration gradient manner, so that when the carrier is
applied to the surface of growth supporting medium, a concentration pattern is transferred
to the surface of the medium fully or partially, which on further processing gives MIC
values based on printed scale on the carrier material.
2. The test device as claimed in claim 1, wherein the porous carrier is a paper test strip.
3. The test device as claimed in claim 1, wherein the chemically or biologically active substance is an antimicrobial agent.
4. The test device as claimed in claim 1, wherein the carrier is treated with a chemical selected from glucose, sodium chloride, cyclodextrins, or hydroxypropylmethyl cellulose.
5. The test device as claimed in claim 1, wherein the porous carrier is used from both sides.
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Abstract
The present invention relates to a test device and a method for the determination of minimum inhibitory concentration of an antimicrobial agent. The invention also relates to producing predefined concentration gradients of chemically and biologically active ingredients on a porous carrier.

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