CA2236552A1 - Determining antimicrobial agent sensitivity for paraffinophilic microorganisms - Google Patents
Determining antimicrobial agent sensitivity for paraffinophilic microorganisms Download PDFInfo
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- CA2236552A1 CA2236552A1 CA 2236552 CA2236552A CA2236552A1 CA 2236552 A1 CA2236552 A1 CA 2236552A1 CA 2236552 CA2236552 CA 2236552 CA 2236552 A CA2236552 A CA 2236552A CA 2236552 A1 CA2236552 A1 CA 2236552A1
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Abstract
A method of determining the sensitivity of at least one paraffinophilic microorganism from a specimen obtained from a patient to different antimicrobial agents and predetermined quantities thereof includes providing at least one receptacle containing an aqueous solution and adjusting the solution to mimic the in vivo clinical conditions of the patient. The method then includes inoculating the solution with the specimen and then placing into the receptacle (i) a paraffin coated slide to bait the at least one paraffinophilic microorganism and (ii) a predetermined quantity of an antimicrobial agent to be tested. The slide is then observed for paraffinophilic microorganism growth or lack thereof to determine whether the predetermined quantity of the antimicrobial agent is effective in inhibiting growth of the paraffinophilic microorganisms on the slide. An associated apparatus is also provided.
Description
CA 02236~2 1998-0~-04 WO97/17424 PCT~S96115317 DETERM]:NIN6 ANTIMICROBIAL AGENT SENSITIVITY FOR PARAFFINOPHILIC
MICROORGANISMS
BACKGROUND OF THF. INVF~TION
This invention relates to a method for determining the antimicrobial agent sensitivity of a paraffinophilic microorganism using various milieus and an associated apparatus, and more particularly, to a method involving the use of a receptacle containing an aqueous solution that mimics the ~n vivo conditions of a patient. A paraffin coated slide is pLaced into the receptacle. The paraffinophilic microor-ganism to be tested for antimicrobial agent sensitivity is baited by the paraffin.
United States Patent Nos. 5,153,119 and 5,316,918, the disclosures of whic:h are incorporated by reference herein, disc:Lose methods and apparatus for identifying and testing the antibiotic sensitivity of Mycobacterium a~ium-intracel 7 u~are ("MAI"). One of the co-inventors herein, Robert-A. Ollar, was the named inventor on these patents. The method of identi-fying MAI includes placing a paraffin coated slide in a receptacle containing a sterile aqueous solution inoculated with a specimen from a patient and analyzing the slide after exposure to the specimen to determine the presence or absence of M~I. The analysis step involves performing a number of speciation assays, such as a tellurite reduction test. The method for testing t:he sensitivity of MAI to different antimicrobial agents amd dosages thereof includes providing a plurality of test tubes adapted to contain an amount of an antimicrobial aqent to be tested and MAI to be assayed and a separate paraffin coated slide adapted for placement in each of t~le test tubes. Observing the growth of MAI on the slide CA 02236~2 1998-0~-04 WO97/17424 PCT~S96/15317 can be used to determine the concentration of the antimicro-bial aqent necessary to resist MAI growth on the slide.
The inventions provide effective, efficient and economical methods for identifying MAI and testing MAI for ~i antimicrobial agent sensitivity. These methods avoid the use of expensive, complicated equipment, and thus can be used in places such as field hospitals and third world locations where the more expensive and hard to use equipment is not available.
Identifying and treating opportunistic infections very often involves educated guesses as to the nature of the microorganism involved and, once identified, the quantity of antimic:robial agent needed to effectively treat the microorganism. Some antimicrobial agents are extremely expensive, so it would be beneficial to use only that amount lS necessary to treat the infection. Furthermore, and more importantly, antimicrobial agents can have undesired side effects, so it is prudent to use only that amount needed to effectively treat the infection. Vnfortunately, however, there is presently no method by which a physician may rapidly ascertain which antimicrobial agent will work best in order to assure effective inhibition of the growth of the microorganism. This has the consequence of greater expense, less efficacy and the potential for more damaging side effect,. This state of affairs exists because medical care givers frequently do not have the type of information regarding antimicrobial agent sensitivity that would make a more exact selection of an antimicrobial agent possible and, once an appropriate antimicrobial agent is selected, facilit;ate a more precise concentration for use. In addition, certain antimicrobial agents react differently under different conditions. For example, one type of antimicrobial agent may not be effective in a low pH environment. It would thus be desirable to mimic the in vivo clinical condition of a patient in vitro in any type of antimicrobial agent sensitivity CA 02236~2 1998-0~-04 WO97/17424 PCT~S96/15317 testing so that antimicrobial agent efficacy against the microoryanism can be maximized.
Thus, there remlains a need for a method of testing the antimicrobial agen,t sensitivity of one or more paraffinophilic microorganisms in a way that maximizes the efficacy of the antimicrobial agent used to inhibit growth of the one or more paraffinophilic microorganisms that may be present in a patient.
SUMMARY OF THF INVF.~TION
The invention has met or surpassed the above-mentioned need as well as others. The method of determining the sensitivity of at least one paraffinophilic microorganism from a specimen obtained from a patient to different antimicrobial agents and predetermined guantities thereof includes providing at least one receptacle containing an aqueous solution and i~djusting the solution to mimic the in vivo clinical conditions of the patient. The method then includes inoculating the solution with the specimen and then placing into the receptacle ~i) a paraffin coated slide to bait the at least one paraffinophilic microorganism and (ii) a predetermined quantity of an antimicrobial agent to be tested.
The slide is then observed for paraffinophilic microorganism growth or lack thereof to determine whether the predetermined quantity of the antimicrobial agent is effective in inhibiting growth of the paraffinophilic microorganisms on the slide.
An associated apparatus is also provided that includes a receptacle adapted to contain an aqueous solution, an amow~t of antimicrobial agent to be tested and the patient specimen and means for adjusting the aqueous solution to mimic the in vivo clinical conditions of the patient. The apparatus further includes a paraffin coated slide adapted to being placed in said receptacle. In this way, observation of the growth of the paraffinophilic microorganisms from the specimen on the slide can be used to determine the concentration of the CA 02236~2 1998-0~-04 W097/17424 PCT~S96/15317 antimic:robial a~ent necessary to resist paraffinophilic microorganism growth on the slide.
BP~F.F DF.SCR~PTION OF THF. DRAW~l~G
A full understanding of the invention can be gained from the following detailed description of the invention when read in conjunction with the accompanying lone drawing which shows one embodiment of the antimicrobial agent sensitivity apparatus.
D~TArT.F.n DFSCRIPTION
lO As used herein, the term "patient" refers to a member of the animal kingdom, including human beings, whose body sp,ecimen is being processed by the method and apparatus of the invention.
As used herein, the term "paraffinophilic" means an organism that can employ paraffin wax as a source of carbon in a basal salt media, devoid of other forms of carbon. The organism can be bacteria:L or funqal in nature.
The method and,apparatus of the invention provide an efficient, effective and economical way of determining the sensitivity of at least one paraffinophilic microorganism to different antimicrobial agents and predetermined quantities thereof. Referring now to the lone Figure, the antimicrobial agent sensitivity method will be explained with reference to one embodiment of the antimicrobial agent sensitivity apparatus 50. The apparatus 50 consists of six receptacles in the form of test tubes 60, 61, 62, 63, 64, 65 each containing an amount of an aqueous solution, such as distilled water 70, 71, 72, 73, 74, 75. It will be appreciated that the aqueous solution should not contain any carbon source, as it is desired to provide a sole carbon source on the slide in order to effe!ctively grow the microorganism to be tested on the slide and not in the aqueous solution. The aqueous solutions CA 02236~2 1998-0~-04 WO97117424 PCT~S96/l5317 contain uniform intervalc; of increasing concentrations of an antimicrobial agent to be tested. For example, the microorganism whose antimicrobial agent sensitivity is to be tested can be MAI, with the antimicrobial agent ciproflaxacin being placed in test tubes 61, 62, 63, 64 and 65 along with aqueous solution 71, 72, 73, 74 and 75. The ciproflaxacin can be placed into each test tube 61, 62, 63, 64 and 65 in increasing concentrations. Test tube 60 is used as a control tube that does not contain any antimicrobial agent.
The specimen from the patient is then inoculated into each of the test tubes 60-65. The specimen can be a blood sample; any biopsy or tissue specimen; stomach fluid;
urine; c:erebral spinal fluid; nasopharyngeal mucosa or saliva.
These s,pecimens can be obtained from the patient in the doctor':3 office or in the emergency room of a hospital, for example, by known techniques.
Slides 80, 81, 82, 83, 84 and 85 coated with paraffin are then placed into respective test tubes 60, 61, 62, 63, 64 and 65. The slides are incubated for a period of eight days. By observing MAI growth 90, 91, 92, 93, 94 on the slides 80-85, the minimum inhibitory concentration ("MIC") of the antimicrobial agent necess~ry to prevent paraffinophilic microorganism growth can be determined. In this case, slide 85 has no MAI growth, thus the MIC concentration is found in test tube 75.
It will be appreciated that although apparatus 50 is shown with multiple receptacles and multiple slides 80-85, that the invention is not limited to multiple receptacles and multiple slides, but covers also a single receptacle and a single slide.
The method of the invention can be used to determine the antibiotic sensitivity of at least one of the paraffinophilic microorganisms selected from the group consist:ing of Micrococcus Paraffinae; Corynebacterium Simplex;
Ahnl; M~cococcus fRhodococcus) CinnabareUsi Ahnl. Mycococcus CA 02236~2 1998-0~-04 WO97/17424 PCT~S96/15317 ~Rhodoc~ Rhodochrous; M~cobact. Perrugosum Var. Athanicum;
Mycobact. ~ubrum Var. P;ropanicum; Mycobacterium Hyalinum;
Mycobaciterium Lacticola; Mycobacterium Album, M. Luteum;
Mycobacterium Microti; Mycobacterium Rubrum, Mycob~cterium Phlei.; Mycobacterium Phlei, M. Smegmatis;
Mycobacterium Testudo; Mycobacterium-Avium-Intracellu~are;
Nocardia Spp.; Actinomyc-es; Candida Lipolytica; Candida Tropica.lis, Torulopsis Colliculosa; Monila Sp., ~n-e6nl~7a Sp., Torula rossa; Penicillium Sp.; I~NL. Aspergillus Flavus;
10 Asperg~llus sp., Penicillium Sp.; Citromyces Sp., Scopulariopsis Sp.; Pseudomonas Fluorescens Liquef~ciens;
Ahnl, P~m. Fluorescens Denitrificans; Pseudomo~as Aeruginosa.
It will be appreciated that in clinical medical practice, there are situations both where a patient has a single paraffinophilic mi.croorganism (such as MAI) or where another patient may have multiple paraffinophilic microorganisms (such as MAI and Mycobacterium Xansasii). It is imperative that all paraffinophilic microorganisms be treated as each one is probably causing pathogenicity in the patient Thus, if an immunocompromised patient has a lung, liver or kidney Ah-C~SS, the physician is interested in the antimicrobial agent that will inhibit all bacterial growth on the slide, whether or not that bacterial growth involves one or mult:iple paraffinophilic microorganisms.
In accordance with the invention, the aqueous solutions 70-75 in each of the test tubes 60-65 can be adjusted to mimic the in vivo "clinical conditions" of the patient By "clinical conditions" it is meant at least one of the follLowing: the pH oI the in vivo milieu of the patient where the paraffinophilic microorganism can be found and the electro:Lyte levels of a patient's blood where paraffinophilic microorc~anism can be found. Adjusting the aqueous solution can be effected by numerous different methods. Adjusting the pH of t:he aqueous solution can be accomplished by adding hydroch:Loric acid (HCl) to obtain a more acidic solution or by CA 02236~2 1998-0~-04 WO97/17424~ PCT~S96115317 adding sodium hydroxide (NaOH) or potassium hydroxide (KOH) in order to obtain a more b~sic solution. Electrolytes such as one or more selected from the group consisting of sodium, potassium, chloride, magnesium, phosphate and calcium, can be added to the solution in desired quantities in order to mimic the electrolytes in the blood of a patient from which a blood sample which may contain the microorganism is obtained.
EXAMPT F. 1 An AIDS patient comes to an emergency room at a hospital complaining of severe abdominal pain probably suffering from an MAI infection and possibly a Mycobacterla Ransasii infection. A gastroenterologist uses a gastrointestinal scope to obtain a specimen of the patient's stomach fluid. The scope indicates that the pH in the patient's stomach is l.5 In the meantime, a lab technician using 1:he apparatus of the Figure adjusts the pH of the aqueous solutions ?0-75 by adding HCl thereto so that the aqueous solutions 70-75 have a pH of l.5. Thus, the pH in the patient's stomach is mimicked by the pH of the aqueous solution in the apparatus shown in the Figure.
After this, the lab technician is instructed by the physician to add specified amounts of an antimicrobial agent to each receptacle 61-65. Receptacle 60 is a control test tube alld thus does not contain any antimicrobial agent.
Receptacle 65 is to contain the highest concentration of antimicrobial agent, with each successive receptacle 64-61 receiving half as much as the adjacent receptacle. In this example, the physician wants to test the sensitivity of the paraffinophilic microorganisms to the antimicrobial agent risabutin. The technician is instructed to add the following amounts to each receptac].e (each receptacle contains 5 cc of aqueous solution) CA 02236~2 1998-0~-04 WO97tl7424 PCT~S96/15317 R~-~.AC$E AMOUNT
61 0.003125 mg 62 0.00625 mg 63 0.0125 mg 64 0.025 mg 0.05 mg After this, portions of the specimen of stomach fluid t:aken by the gast:roenterologist from the patient are inocula.ted into each of the receptacles 60-65 holding a respective paraffin coated slide 80-85. After about eight d.ays the growth or lack thereof is observed to indicate which c:oncentration of antimicrobial agent is effective to inhibit paraffinophilic microorganism growth on the slide.
EXAMP~ F 2 An AIDS patient comes to an emergency room complaining of high fever and apparently has pneumonia. The physician suspects that there is an infection caused by Nocardia bactereremia. As is standard in almost every emergency room, a chemical screen ~"CSS") is performed on a blood specimen obtained :Erom the patient. The CSS lists the electrolyte content of the patient's blood. The electrolyte content is communicated to a lab technician who in turn adjusts the aqueous solutions 70-75 in the receptacles 60-65 each holding a respective paraffin coated slide 80-85. For example, the CSS reveals that the patient has a sodium level of 120. The lab technician adjusts the sodium level of the aqueous solution (for e.xample, distilled water) by adding sodium thereto in order to mimic the 120 level of sodium found in the patient's blood.
CA 02236~2 1998-0~-04 After this, the lab technician is instructed by the physician to add specified amounts of an antimicrobial agent to each receptacle 61-65. Receptacle 60 is a control test tube and thus does not contain any antimicrobial agent.
5 Receptarle 65 is to contain the highest concentration of antimic:robial agent, with each successive receptacle 64-61 receiving half as much as the adjacent receptacle. In this example, the physician wants to test the sensitivity of the paraffinophilic microorganisms to the antimicrobial agent lO clarithromycin. The technician is instructed to add the following amounts to each receptacle (each receptacle contalns cc of a~ueous solution) a~ .AcL~ AMo~
61 0.00625 mg 62 0.0125 mg 63 0.025 mg 64 0.05 mg O.l mg After this, a portion of the blood specimen taken from th,e patient are inoculated into each receptacle 60-65 holding a respective paraffin coated slide 80-85. After about eight days the growth or lac~c thereof is observed to indicate 25 which concentration of antimicrobial agent is effective to inhibit paraffinophilic microorganism growth on the slide.
It will be appreciated that a method for determining the antimicrobial agent sensitivity of a microorganism is providecl. The method includes adjusting the aqueous solutions 30 used in the testing appar-atus to mimic the in vivo clinical conditions of a patient from whom the specimen containing the microorqanism to be identified and tested is obtained. The method ia effective and efficient and do not involve the use CA 02236~2 1998-0~-04 2'1 PCT~S96/15317 of expensive and comp:Licated equipment. An associated apparat:us is also disclosed.
While specific embodiments of the invention have been disclosed, it will be appreciated by those skilled in the art that various modifications and alterations to those details could be developed in light of the overall teachings of the disclosure. Accordingly, the particular arrangements disclosed are meant to be illustrative only and not limiting as to the scope of the invention which is to be given the full breadth of the appended claims and any and all equivalents thereof.
MICROORGANISMS
BACKGROUND OF THF. INVF~TION
This invention relates to a method for determining the antimicrobial agent sensitivity of a paraffinophilic microorganism using various milieus and an associated apparatus, and more particularly, to a method involving the use of a receptacle containing an aqueous solution that mimics the ~n vivo conditions of a patient. A paraffin coated slide is pLaced into the receptacle. The paraffinophilic microor-ganism to be tested for antimicrobial agent sensitivity is baited by the paraffin.
United States Patent Nos. 5,153,119 and 5,316,918, the disclosures of whic:h are incorporated by reference herein, disc:Lose methods and apparatus for identifying and testing the antibiotic sensitivity of Mycobacterium a~ium-intracel 7 u~are ("MAI"). One of the co-inventors herein, Robert-A. Ollar, was the named inventor on these patents. The method of identi-fying MAI includes placing a paraffin coated slide in a receptacle containing a sterile aqueous solution inoculated with a specimen from a patient and analyzing the slide after exposure to the specimen to determine the presence or absence of M~I. The analysis step involves performing a number of speciation assays, such as a tellurite reduction test. The method for testing t:he sensitivity of MAI to different antimicrobial agents amd dosages thereof includes providing a plurality of test tubes adapted to contain an amount of an antimicrobial aqent to be tested and MAI to be assayed and a separate paraffin coated slide adapted for placement in each of t~le test tubes. Observing the growth of MAI on the slide CA 02236~2 1998-0~-04 WO97/17424 PCT~S96/15317 can be used to determine the concentration of the antimicro-bial aqent necessary to resist MAI growth on the slide.
The inventions provide effective, efficient and economical methods for identifying MAI and testing MAI for ~i antimicrobial agent sensitivity. These methods avoid the use of expensive, complicated equipment, and thus can be used in places such as field hospitals and third world locations where the more expensive and hard to use equipment is not available.
Identifying and treating opportunistic infections very often involves educated guesses as to the nature of the microorganism involved and, once identified, the quantity of antimic:robial agent needed to effectively treat the microorganism. Some antimicrobial agents are extremely expensive, so it would be beneficial to use only that amount lS necessary to treat the infection. Furthermore, and more importantly, antimicrobial agents can have undesired side effects, so it is prudent to use only that amount needed to effectively treat the infection. Vnfortunately, however, there is presently no method by which a physician may rapidly ascertain which antimicrobial agent will work best in order to assure effective inhibition of the growth of the microorganism. This has the consequence of greater expense, less efficacy and the potential for more damaging side effect,. This state of affairs exists because medical care givers frequently do not have the type of information regarding antimicrobial agent sensitivity that would make a more exact selection of an antimicrobial agent possible and, once an appropriate antimicrobial agent is selected, facilit;ate a more precise concentration for use. In addition, certain antimicrobial agents react differently under different conditions. For example, one type of antimicrobial agent may not be effective in a low pH environment. It would thus be desirable to mimic the in vivo clinical condition of a patient in vitro in any type of antimicrobial agent sensitivity CA 02236~2 1998-0~-04 WO97/17424 PCT~S96/15317 testing so that antimicrobial agent efficacy against the microoryanism can be maximized.
Thus, there remlains a need for a method of testing the antimicrobial agen,t sensitivity of one or more paraffinophilic microorganisms in a way that maximizes the efficacy of the antimicrobial agent used to inhibit growth of the one or more paraffinophilic microorganisms that may be present in a patient.
SUMMARY OF THF INVF.~TION
The invention has met or surpassed the above-mentioned need as well as others. The method of determining the sensitivity of at least one paraffinophilic microorganism from a specimen obtained from a patient to different antimicrobial agents and predetermined guantities thereof includes providing at least one receptacle containing an aqueous solution and i~djusting the solution to mimic the in vivo clinical conditions of the patient. The method then includes inoculating the solution with the specimen and then placing into the receptacle ~i) a paraffin coated slide to bait the at least one paraffinophilic microorganism and (ii) a predetermined quantity of an antimicrobial agent to be tested.
The slide is then observed for paraffinophilic microorganism growth or lack thereof to determine whether the predetermined quantity of the antimicrobial agent is effective in inhibiting growth of the paraffinophilic microorganisms on the slide.
An associated apparatus is also provided that includes a receptacle adapted to contain an aqueous solution, an amow~t of antimicrobial agent to be tested and the patient specimen and means for adjusting the aqueous solution to mimic the in vivo clinical conditions of the patient. The apparatus further includes a paraffin coated slide adapted to being placed in said receptacle. In this way, observation of the growth of the paraffinophilic microorganisms from the specimen on the slide can be used to determine the concentration of the CA 02236~2 1998-0~-04 W097/17424 PCT~S96/15317 antimic:robial a~ent necessary to resist paraffinophilic microorganism growth on the slide.
BP~F.F DF.SCR~PTION OF THF. DRAW~l~G
A full understanding of the invention can be gained from the following detailed description of the invention when read in conjunction with the accompanying lone drawing which shows one embodiment of the antimicrobial agent sensitivity apparatus.
D~TArT.F.n DFSCRIPTION
lO As used herein, the term "patient" refers to a member of the animal kingdom, including human beings, whose body sp,ecimen is being processed by the method and apparatus of the invention.
As used herein, the term "paraffinophilic" means an organism that can employ paraffin wax as a source of carbon in a basal salt media, devoid of other forms of carbon. The organism can be bacteria:L or funqal in nature.
The method and,apparatus of the invention provide an efficient, effective and economical way of determining the sensitivity of at least one paraffinophilic microorganism to different antimicrobial agents and predetermined quantities thereof. Referring now to the lone Figure, the antimicrobial agent sensitivity method will be explained with reference to one embodiment of the antimicrobial agent sensitivity apparatus 50. The apparatus 50 consists of six receptacles in the form of test tubes 60, 61, 62, 63, 64, 65 each containing an amount of an aqueous solution, such as distilled water 70, 71, 72, 73, 74, 75. It will be appreciated that the aqueous solution should not contain any carbon source, as it is desired to provide a sole carbon source on the slide in order to effe!ctively grow the microorganism to be tested on the slide and not in the aqueous solution. The aqueous solutions CA 02236~2 1998-0~-04 WO97117424 PCT~S96/l5317 contain uniform intervalc; of increasing concentrations of an antimicrobial agent to be tested. For example, the microorganism whose antimicrobial agent sensitivity is to be tested can be MAI, with the antimicrobial agent ciproflaxacin being placed in test tubes 61, 62, 63, 64 and 65 along with aqueous solution 71, 72, 73, 74 and 75. The ciproflaxacin can be placed into each test tube 61, 62, 63, 64 and 65 in increasing concentrations. Test tube 60 is used as a control tube that does not contain any antimicrobial agent.
The specimen from the patient is then inoculated into each of the test tubes 60-65. The specimen can be a blood sample; any biopsy or tissue specimen; stomach fluid;
urine; c:erebral spinal fluid; nasopharyngeal mucosa or saliva.
These s,pecimens can be obtained from the patient in the doctor':3 office or in the emergency room of a hospital, for example, by known techniques.
Slides 80, 81, 82, 83, 84 and 85 coated with paraffin are then placed into respective test tubes 60, 61, 62, 63, 64 and 65. The slides are incubated for a period of eight days. By observing MAI growth 90, 91, 92, 93, 94 on the slides 80-85, the minimum inhibitory concentration ("MIC") of the antimicrobial agent necess~ry to prevent paraffinophilic microorganism growth can be determined. In this case, slide 85 has no MAI growth, thus the MIC concentration is found in test tube 75.
It will be appreciated that although apparatus 50 is shown with multiple receptacles and multiple slides 80-85, that the invention is not limited to multiple receptacles and multiple slides, but covers also a single receptacle and a single slide.
The method of the invention can be used to determine the antibiotic sensitivity of at least one of the paraffinophilic microorganisms selected from the group consist:ing of Micrococcus Paraffinae; Corynebacterium Simplex;
Ahnl; M~cococcus fRhodococcus) CinnabareUsi Ahnl. Mycococcus CA 02236~2 1998-0~-04 WO97/17424 PCT~S96/15317 ~Rhodoc~ Rhodochrous; M~cobact. Perrugosum Var. Athanicum;
Mycobact. ~ubrum Var. P;ropanicum; Mycobacterium Hyalinum;
Mycobaciterium Lacticola; Mycobacterium Album, M. Luteum;
Mycobacterium Microti; Mycobacterium Rubrum, Mycob~cterium Phlei.; Mycobacterium Phlei, M. Smegmatis;
Mycobacterium Testudo; Mycobacterium-Avium-Intracellu~are;
Nocardia Spp.; Actinomyc-es; Candida Lipolytica; Candida Tropica.lis, Torulopsis Colliculosa; Monila Sp., ~n-e6nl~7a Sp., Torula rossa; Penicillium Sp.; I~NL. Aspergillus Flavus;
10 Asperg~llus sp., Penicillium Sp.; Citromyces Sp., Scopulariopsis Sp.; Pseudomonas Fluorescens Liquef~ciens;
Ahnl, P~m. Fluorescens Denitrificans; Pseudomo~as Aeruginosa.
It will be appreciated that in clinical medical practice, there are situations both where a patient has a single paraffinophilic mi.croorganism (such as MAI) or where another patient may have multiple paraffinophilic microorganisms (such as MAI and Mycobacterium Xansasii). It is imperative that all paraffinophilic microorganisms be treated as each one is probably causing pathogenicity in the patient Thus, if an immunocompromised patient has a lung, liver or kidney Ah-C~SS, the physician is interested in the antimicrobial agent that will inhibit all bacterial growth on the slide, whether or not that bacterial growth involves one or mult:iple paraffinophilic microorganisms.
In accordance with the invention, the aqueous solutions 70-75 in each of the test tubes 60-65 can be adjusted to mimic the in vivo "clinical conditions" of the patient By "clinical conditions" it is meant at least one of the follLowing: the pH oI the in vivo milieu of the patient where the paraffinophilic microorganism can be found and the electro:Lyte levels of a patient's blood where paraffinophilic microorc~anism can be found. Adjusting the aqueous solution can be effected by numerous different methods. Adjusting the pH of t:he aqueous solution can be accomplished by adding hydroch:Loric acid (HCl) to obtain a more acidic solution or by CA 02236~2 1998-0~-04 WO97/17424~ PCT~S96115317 adding sodium hydroxide (NaOH) or potassium hydroxide (KOH) in order to obtain a more b~sic solution. Electrolytes such as one or more selected from the group consisting of sodium, potassium, chloride, magnesium, phosphate and calcium, can be added to the solution in desired quantities in order to mimic the electrolytes in the blood of a patient from which a blood sample which may contain the microorganism is obtained.
EXAMPT F. 1 An AIDS patient comes to an emergency room at a hospital complaining of severe abdominal pain probably suffering from an MAI infection and possibly a Mycobacterla Ransasii infection. A gastroenterologist uses a gastrointestinal scope to obtain a specimen of the patient's stomach fluid. The scope indicates that the pH in the patient's stomach is l.5 In the meantime, a lab technician using 1:he apparatus of the Figure adjusts the pH of the aqueous solutions ?0-75 by adding HCl thereto so that the aqueous solutions 70-75 have a pH of l.5. Thus, the pH in the patient's stomach is mimicked by the pH of the aqueous solution in the apparatus shown in the Figure.
After this, the lab technician is instructed by the physician to add specified amounts of an antimicrobial agent to each receptacle 61-65. Receptacle 60 is a control test tube alld thus does not contain any antimicrobial agent.
Receptacle 65 is to contain the highest concentration of antimicrobial agent, with each successive receptacle 64-61 receiving half as much as the adjacent receptacle. In this example, the physician wants to test the sensitivity of the paraffinophilic microorganisms to the antimicrobial agent risabutin. The technician is instructed to add the following amounts to each receptac].e (each receptacle contains 5 cc of aqueous solution) CA 02236~2 1998-0~-04 WO97tl7424 PCT~S96/15317 R~-~.AC$E AMOUNT
61 0.003125 mg 62 0.00625 mg 63 0.0125 mg 64 0.025 mg 0.05 mg After this, portions of the specimen of stomach fluid t:aken by the gast:roenterologist from the patient are inocula.ted into each of the receptacles 60-65 holding a respective paraffin coated slide 80-85. After about eight d.ays the growth or lack thereof is observed to indicate which c:oncentration of antimicrobial agent is effective to inhibit paraffinophilic microorganism growth on the slide.
EXAMP~ F 2 An AIDS patient comes to an emergency room complaining of high fever and apparently has pneumonia. The physician suspects that there is an infection caused by Nocardia bactereremia. As is standard in almost every emergency room, a chemical screen ~"CSS") is performed on a blood specimen obtained :Erom the patient. The CSS lists the electrolyte content of the patient's blood. The electrolyte content is communicated to a lab technician who in turn adjusts the aqueous solutions 70-75 in the receptacles 60-65 each holding a respective paraffin coated slide 80-85. For example, the CSS reveals that the patient has a sodium level of 120. The lab technician adjusts the sodium level of the aqueous solution (for e.xample, distilled water) by adding sodium thereto in order to mimic the 120 level of sodium found in the patient's blood.
CA 02236~2 1998-0~-04 After this, the lab technician is instructed by the physician to add specified amounts of an antimicrobial agent to each receptacle 61-65. Receptacle 60 is a control test tube and thus does not contain any antimicrobial agent.
5 Receptarle 65 is to contain the highest concentration of antimic:robial agent, with each successive receptacle 64-61 receiving half as much as the adjacent receptacle. In this example, the physician wants to test the sensitivity of the paraffinophilic microorganisms to the antimicrobial agent lO clarithromycin. The technician is instructed to add the following amounts to each receptacle (each receptacle contalns cc of a~ueous solution) a~ .AcL~ AMo~
61 0.00625 mg 62 0.0125 mg 63 0.025 mg 64 0.05 mg O.l mg After this, a portion of the blood specimen taken from th,e patient are inoculated into each receptacle 60-65 holding a respective paraffin coated slide 80-85. After about eight days the growth or lac~c thereof is observed to indicate 25 which concentration of antimicrobial agent is effective to inhibit paraffinophilic microorganism growth on the slide.
It will be appreciated that a method for determining the antimicrobial agent sensitivity of a microorganism is providecl. The method includes adjusting the aqueous solutions 30 used in the testing appar-atus to mimic the in vivo clinical conditions of a patient from whom the specimen containing the microorqanism to be identified and tested is obtained. The method ia effective and efficient and do not involve the use CA 02236~2 1998-0~-04 2'1 PCT~S96/15317 of expensive and comp:Licated equipment. An associated apparat:us is also disclosed.
While specific embodiments of the invention have been disclosed, it will be appreciated by those skilled in the art that various modifications and alterations to those details could be developed in light of the overall teachings of the disclosure. Accordingly, the particular arrangements disclosed are meant to be illustrative only and not limiting as to the scope of the invention which is to be given the full breadth of the appended claims and any and all equivalents thereof.
Claims (15)
1. A method of determining the sensitivity of at least one paraffinophilic microorganism from a specimen obtained from a patient having in vivo clinical conditions to different antimicrobial agents and predetermined quantities thereof, said method comprising:
providing at least one receptacle containing an aqueous solution;
adjusting said solution to mimic said in vivo clinical conditions of said patient;
inoculating said solution with said specimen;
placing into said receptacle (i) a paraffin coated slide to bait said paraffinophilic microorganism and (ii) a predetermined quantity of an antimicrobial agent to be tested; and observing said paraffinophilic microorganism growth or lack thereof on said slide to determine whether said predetermined quantity of said antimicrobial agent is effective in inhibiting growth of said paraffinophilic microorganism on said slide.
providing at least one receptacle containing an aqueous solution;
adjusting said solution to mimic said in vivo clinical conditions of said patient;
inoculating said solution with said specimen;
placing into said receptacle (i) a paraffin coated slide to bait said paraffinophilic microorganism and (ii) a predetermined quantity of an antimicrobial agent to be tested; and observing said paraffinophilic microorganism growth or lack thereof on said slide to determine whether said predetermined quantity of said antimicrobial agent is effective in inhibiting growth of said paraffinophilic microorganism on said slide.
2. The method of Claim 1, including determining a pH of said specimen; and adjusting said aqueous solution to have a pH
generally equal to said pH of said specimen.
generally equal to said pH of said specimen.
3. The method of Claim 2, including altering said pH of said aqueous solution by adding a chemical to said aqueous solution.
4. The method of Claim 3, including said chemical is selected from the group consisting of HCl, KOH and NaOH.
5. The method of Claim 1, including employing as said specimen a blood sample of said patient;
determining an electrolyte level of said blood sample; and adjusting said solution to have an electrolyte level generally equal to said electrolyte level of said patient's blood.
determining an electrolyte level of said blood sample; and adjusting said solution to have an electrolyte level generally equal to said electrolyte level of said patient's blood.
6. The method of Claim 5, including adjusting said aqueous solution by adding thereto at least one of the group consisting of sodium, potassium, chloride, magnesium, phosphate and calcium.
7. The method of Claim 1, including employing said method to determine the antibiotic sensitivity of at least one member of the group consisting of Micrococcus Paraffinae; Corynebacterium Simplex;
Ahnl; Mycococcus (Rhodococcus) Cinnabareus; Ahnl. Mycococcus (Rhodoc) Rhodochrous; Mycobact. Perrugosum Var. Athanicum;
Mycobact. Rubrum Var. Propanicum; Mycobacterium Hyalinum;
Mycobacterium Lacticola; Mycobacterium Album, M. Luteum;
Mycobacterium Microti; Mycobacterium Rubrum, Mycobacterium Phlei.; Mycobacterium Phlei, M. Smegmatis;
Mycobacterium Testudo; Mycobacterium-Avium-Intracellulare;
Nocardia Spp.; Actinomyces; Candida Lipolytica; Candida Tropicalis, Torulopsis Colliculosa; Monila Sp., Hansenula Sp., Torula rossa; Penicillium Sp.; IHNL . Aspergillus Flavus;
Aspergillus sp., Penicillium Sp.; Citromyces Sp., Scopulariopsis Sp.; Pseudomonas Fluorescens Liquefaciens;
Ahnl, Pem. Fluorescens Denitrificans; Pseudomonas Aeruginosa.
Ahnl; Mycococcus (Rhodococcus) Cinnabareus; Ahnl. Mycococcus (Rhodoc) Rhodochrous; Mycobact. Perrugosum Var. Athanicum;
Mycobact. Rubrum Var. Propanicum; Mycobacterium Hyalinum;
Mycobacterium Lacticola; Mycobacterium Album, M. Luteum;
Mycobacterium Microti; Mycobacterium Rubrum, Mycobacterium Phlei.; Mycobacterium Phlei, M. Smegmatis;
Mycobacterium Testudo; Mycobacterium-Avium-Intracellulare;
Nocardia Spp.; Actinomyces; Candida Lipolytica; Candida Tropicalis, Torulopsis Colliculosa; Monila Sp., Hansenula Sp., Torula rossa; Penicillium Sp.; IHNL . Aspergillus Flavus;
Aspergillus sp., Penicillium Sp.; Citromyces Sp., Scopulariopsis Sp.; Pseudomonas Fluorescens Liquefaciens;
Ahnl, Pem. Fluorescens Denitrificans; Pseudomonas Aeruginosa.
8. The method of Claim 1, including providing a plurality of receptacles each containing an aqueous solution;
inoculating each receptacle with an amount of said specimen;
placing (i) a separate paraffin coated slide and (ii) an amount of an antimicrobial agent to be tested in each receptacle, each said receptacle containing a different predetermined quantity of said antimicrobial agent; and observing said paraffinophilic microorganism growth or lack thereof on said slides to determine the minimum inhibitory concentration of said antimicrobial agent to inhibit growth of said paraffinophilic microorganism.
inoculating each receptacle with an amount of said specimen;
placing (i) a separate paraffin coated slide and (ii) an amount of an antimicrobial agent to be tested in each receptacle, each said receptacle containing a different predetermined quantity of said antimicrobial agent; and observing said paraffinophilic microorganism growth or lack thereof on said slides to determine the minimum inhibitory concentration of said antimicrobial agent to inhibit growth of said paraffinophilic microorganism.
9. The method of Claim 1, including before adjusting said aqueous solution, testing said specimen to determine said clinical condition of said patient.
10. An apparatus for determining the sensitivity of at least one paraffinophilic microorganism from a specimen obtained from a patient having in vivo clinical conditions to different antimicrobial agents and predetermined quantities thereof comprising:
a receptacle adapted to contain an aqueous solution, an amount of antimicrobial agent to be tested and said specimen;
means for adjusting said aqueous solution to mimic said in vivo clinical conditions of said patient; and a paraffin coated slide adapted to being placed in said receptacle.
a receptacle adapted to contain an aqueous solution, an amount of antimicrobial agent to be tested and said specimen;
means for adjusting said aqueous solution to mimic said in vivo clinical conditions of said patient; and a paraffin coated slide adapted to being placed in said receptacle.
11. The apparatus of Claim 10, wherein said means for adjusting includes chemicals to adjust the pH of said aqueous solution.
12. The apparatus of Claim 11, wherein said chemicals are selected from the group consisting of HC1, KOH and NaOH.
13. The apparatus of Claim 10, wherein said adjusting means includes chemicals to mimic the electrolyte level in said patient's blood.
14. The apparatus of Claim 13, wherein said chemicals include at least one of the group consisting of sodium, potassium, chloride, magnesium, phosphate and calcium.
15. The apparatus of Claim 10, including a plurality of receptacles each adapted to contain an aqueous solution, an amount of antimicrobial agent to be tested and said specimen; and a plurality of paraffin coated slides each of which is adapted to being placed in one of said receptacles.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US55573695A | 1995-11-09 | 1995-11-09 | |
| US08/555,736 | 1995-11-09 | ||
| PCT/US1996/015317 WO1997017424A1 (en) | 1995-11-09 | 1996-09-25 | Determining antimicrobial agent sensitivity for paraffinophilic microorganisms |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2236552A1 true CA2236552A1 (en) | 1997-05-15 |
Family
ID=29405945
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2236552 Abandoned CA2236552A1 (en) | 1995-11-09 | 1996-09-25 | Determining antimicrobial agent sensitivity for paraffinophilic microorganisms |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA2236552A1 (en) |
-
1996
- 1996-09-25 CA CA 2236552 patent/CA2236552A1/en not_active Abandoned
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