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  • 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
  • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor

Definitions

• This invention relates to a method for speciating and identifying paraffinophilic materials.
• HIV Human immunodeficiency virus type 1 or HIV causes acquired immunodeficiency syndrome ("AIDS") which is a fatal disease approaching epidemic proportions throughout the world. By current estimates, 110 million people will be infected with HIV by the year 2010. When AIDS develops, it is usually characterized by opportunistic infection, such as Pneumocystic pneumonia, Kaposi's sarcoma, lymphoma and Mvcobacterium avium intracellulare complex (MAI) .
• opportunistic infection such as Pneumocystic pneumonia, Kaposi's sarcoma, lymphoma and Mvcobacterium avium intracellulare complex (MAI) .
• Organisms of MAI prior to the AIDS epidemic were recognized as a rare form of pneumonia in patients with chronic lung infections (E. Wolinsky, Nontuberculous mycobacteria and associated diseases. Am. Rev. Respir. Dis. 1979, 119:107-59). Organisms of MAI comprise two closely related species, M. avium and M. intracellulare. which have minor virulence in the non-HIV host. By 1980, only 24 cases of MAI had been reported in the medical literature (C. R. Horsburgh, Jr. et al., Disseminated infection with Mycobacterium avium intracellulare. Medicine (Baltimore) 1985, 64:36-48). However, the epidemic of disseminated MAI infection is concurrent with the AIDS epidemic.
• Carbon-14 labelled palmitic acid In use, vials containing mycobacterial growth give off Carbon-14 labelled C0 2 and this is detected by a device similar to that used for liquid scintillation capable of detecting beta emitters.
• Another method of isolation in blood involves using genetic probes which rely upon DNA hybridization (C. M. Reichert et al., Pathologic features of AIDS. In: V. T. DeVita Jr. et al. (eds) AIDS etiology, diagnosis, treatment and prevention, p. 134 NY J. 13, Lipponcott, 1985).
• 5,153,119 which names one of the joint inventors of the present invention as sole inventor, discloses a method for speciating and identifying MAI in a specimen and involves the use of paraffin coated slides to determine the presence or absence of atypical Mycobacteria (mycobacteria other than M. tuberculosis. M. laprae. and M. paratuberculosis) .
• This process while quite effective for isolating and speciating of MAI, may for some purposes be deemed to relatively slow, taking on the order of about 6 days (in feces) to 34.5 days (in blood).
• the disclosure of this patent is expressly incorporated herein by reference.
• 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 may be bacterial or fungal in nature.
• the present invention has met the above described need. It provides a method of determining the presence of a paraffinophilic organism in a body specimen by introducing portions of the specimen into a plurality of receptacles which contain a sterile broth and antibiotics. One paraffin coated slide is introduced into each receptacle with the slides being observed for the presence of organisms growing thereon. After observing such growth, a first slide is subjected to an alcohol-acid fastness test.
• a second slide is subjected to a tellurite reduction assay to determine the possibility of the presence of a paraffinophilic organism on the second slide. If it is determined that there is a possibility of the presence of a paraffinophilic organism on the second slide, a third slide is subjected to at least one speciation assay to confirm the presence of a paraffinophilic organism on the slide. Subsequently, a DNA extraction is employed on at least one additional slide to determine whether a paraffinophilic organism is present on the specimen.
• Figure 1 is a schematic front elevational view of a receptacle holding a paraffin coated slide in a sterile aqueous solution containing the fecal specimen.
• Figure 2 is a schematic illustration of the acid- alcohol fastness assay employable in the present invention.
• Figure 3 is a schematic illustration of a tellurite reduction assay.
• Figure 4 is a schematic illustration of a nitrate reduction assay.
• Figure 5 is a schematic illustration of a urea hydrolysis assay.
• FIG. 6 is a schematic illustration of a Tween 80 hydrolysis.
• FIG. 7 is a schematic illustration of the use of DNA extraction in accordance with the present invention.
• the term "patient” refers to a member of the animal kingdom, including human beings, whose body specimen is being processed by the system of the present invention.
• body specimen shall include fecal matter, blood, sputum, tissue, and cerebral spinal fluid obtained from a patient.
• paraffinophilic shall expressly include, but not be limited to the following organisms: Micrococcus Paraffinae; Corynebacterium Simplex;
• Rhodoc Rhodochrous
• Mycobact Perru ⁇ osum Var. Athanicum
• Actinomvces Candida Lipolytica; Candida Tropicalis. Torulopsis Colliculosa: Monilia Sp.. Hansenula Sp.. Torula rossa; Penicillium Sp. ; IHNL. Asperqillus Flavus:
• Scopulariopsis Sp. Pseudomonas Fluorescens Liquefaciens; Ahnl, Pem. Fluorescens Denitrificans; Pseudomonas Aeru inosa.
• Paraffinophilic organism related opportunistic pathology may be detected by the present invention. For example, it is known that Hodgkin's disease has an adverse effect on cellular immunity, as does intentional immunosuppression employed in cellular, tissue, or organ transplants.
• MAI in fecal matter is advantageous in accomplishing this objective as MAI is initially found in the gastrointestinal tract (E. C. Klatt et al.. Pathology of Mycobacterium avium-intracellulare infection in acquired immunodeficiency syndrome; Hum. Pathol. 1987; 18: 709-14). Colonization of the gastrointestinal tract precedes the ability to isolate MAI in the blood by many months and thereby offers an advantageous means for getting an early warning. Gut histopathology of AIDS patients reveals a large number of acid-fast bacilli in both the mucosal and sub ucosal layers of the gastrointestinal tract.
• the localized gastrointestinal infection occurs most commonly in the duodenum causing a lymphadenopathy. Additional areas of MAI infection are the terminal ileum and appendix. The increased number of Payer's patches increases local MAI concentrations. Ethanol ingestion is associated with a significant increase in MAI infection of the gastrointestinal tract. This is believed to be due to the enhanced passage of MAI across the intestinal wall secondary to an alcohol induced enteritis (L. E. Bermudez et al.. An animal model of Mycobacterium avium complex disseminated infection after colonization of the intestinal tract; Kuzell Institute for Arthritis and Infectious Diseases; Medical Research Institute of San Francisco at California Pacific Medical Center 94115; J. Infect. Dis. 1992 Jan.; 165(1): 75-9) . Symptoms involved in gastrointestinal traction infection are nausea, diarrhea, abdominal pain, biliary obstruction and severe cachexia.
• the colonization of MAI of the gastrointestinal tract may be due to transmission through the water supply.
• Studies from Jefferson Medical College have documented colonization of that hospital's hot water system beginning in 1982. It was also found that colonization of normal volunteers could be demonstrated after they gargled with water from the hospital (S. A. Murphy et al., Mycobacterium avium intracellulare in a hospital hot water system: epidemio- logical investigation; In: Proceedings and abstracts of the 24th Interscience Conference on Antimicrobial Agents and Chemotherapy, Las Vegas, Oct. 24-26, 1983, Washington, D.C.; American Society for Microbiology, 1983; 277) .
• the home water supply of HIV patients may be the potentially infectious source of initial transmission of MAI.
• the HIV wasting syndrome is a clinically defined entity in which findings of profound involuntary weight loss greater than 10% of baseline body weight plus either chronic diarrhea (at least two loose stools per day for greater than or equal to 30 days) or chronic weakness and documented fever (for greater than or equal to 30 days, intermittent or constant) in the absence of concurrent illness or condition other than HIV infection that could explain the findings such as cancer, cryptosporidiosis, or other specific enteritis, for example, (P. Ma et al. , AIDS and Infections of Homosexual Men
• the present invention includes an adaptation of the MAI Para SL/C method which we have designated as the MAI ParaPecogen method. The latter has been specifically designed for periodic testing of ARC patients and provides quick and inexpensive means of early detection of the presence of gastrointestinal in accordance with the above criteria.
• a suspension may be prepared by suspending a 4mm loopful of fresh body specimen in 3 to 5 milliliters of sterile saline.
• a kit 10 a 0.5 milliliter specimen may be, as shown in Figure 1, introduced into a test tube 12 which contains a sterile aqueous solution 14 (such as a Czapek broth) and a cotton plug 16 to seal the test tube.
• the specimen to be tested for the presence or absence of paraffinophilic organisms may be introduced into the test tube 12 and a paraffin coated slide 18 is subsequently analyzed.
• the paraffin coated slides may be made in accordance with the teachings of Ollar, United States Patent No. 5,153,119.
• the Czapek broth 14 may be provided with an anti-bacterial, an anti-fungal/antibiotic cocktail such as that sold under the trade name "PANTA” sold by Becton, Dickenson/Johnston Labs Division. This product tends to resist possible contaminating factors such as Pseudomonas aeruqinosa or Candida tropicalis. This product will have no effect on the paraffinophilic organism MAI which is resistant to the currently used antibiotic in "PANTA.”
• the kit 10 can also serve as a means of distinguishing between atypical mycobacteria and nocardioform organisms on the one hand and mycobacteria tuberculosis on the other hand because the latter cannot utilize paraffin was as a sole source of carbon.
• a tropism is created between the paraffin and organisms capable of using the paraffin as its carbon source, such as atypical paraffinophilic mycobacteria and paraffinophilic nocardioform organisms.
• the outward manifestation of this tropism or baiting is the appearance of growth on the paraffin surface. Paraffinophilic mycobacterial or nocardial presence on the slide is determined by an alcohol-acid fastness test 40 ( Figure 2) .
• This test can be used to further distinguish between the atypical mycobacteria and the nocardioform organisms.
• atypical paraffinophilic mycobacteria are alcohol-acid fast; paraffinophilic nocardioform organisms are acid-fast and paraffinophilic Pseudomonas aeruginosa or paraffinophilic Candida tropicalis are neither acid nor alcohol-acid fast.
• these latter two paraffinophilic groups can be eliminated as possibilities by the alcohol- acid fastness testing.
• test tubes 50, 51, 52, 53, 54, 55, 56, 57 are each provided with about 4.5 milliliters of sterile Czapek broth containing the antibacterial and anti- fungal/antibiotic cocktail as well as 0.5 ml of the fresh fecal suspension.
• a paraffin coated slide is then introduced into each of the body specimen inoculated Czapek broth antibiotic tubes 50-57 in a manner to be described hereinafter.
• the alcohol-acid fastness testing means 40 of Figure 2 results in the solution staining the paraffinophilic organisms on the slide for subsequent analysis under a microscope.
• the receptacles or tubes 50-57 are incubated at about 37°C.
• one of the slides is removed a tube and stained with Kinyoun Acid-alcohol stain as disclosed in United States Patent No. 5,153,119.
• Another slide is withdrawn and subcultured in Lowenstein-Jensen media as disclosed in United States Patent No. 5,153,119.
• a third slide is removed and tested for tellurite reduction as disclosed in United States Patent No. 5,153,119.
• a further slide is removed and testing for nitrate reduction in accordance with United States Patent No. 5,153,119.
• a further slide is removed and tested for urea hydrolysis in accordance with United States Patent No. 5,153,119.
• Another slide is removed and tested for Tween 80 hydrolysis as disclosed in United States Patent No. 5,153,119.
• the paraffin coated slide culture with visible paraffinophilic organism growth 42 is removed from the test tube 12 of Figure 1 and is first immersed in two consecutive tubes 50, 51 of distilled water and then immersed in a tube 52 of Kinyoun carbolfuchsin for fifteen minutes.
• the slide 42 is again immersed in a tube 53 of distilled water and then placed in a tube 54 containing acid-alcohol consisting of 97 ml absolute ethanol and 3.0 ml concentrated HC1 for five minutes.
• the slide is washed in a fourth tube 55 of distilled water and then placed into a tube 56 of 1.0% (v/v) aqueous Methylene blue solution for 1 minute.
• the slide is washed in a fifth tube 57 of distilled water.
• the slide culture is then removed from the fifth tube 57 of distilled water and blotted gently with a clean absorbent paper tissue.
• the slide culture is then viewed under a microscope at 250x, 450x and lOOOx oil immersion.
• Figure 3 shows the tellurite reduction assay which consists of a test tube 60 filled, preferably, with a Czapek broth plus an amount of potassium tellurite reagent 62.
• a cultured slide 43 is immersed into the test tube 60 and incubated. If a paraffinophilic MAI organism is present on the slide, a heavy black precipitate 64 forms at the level of the meniscus pellicle 65 of the slide 43. This test alerts the user to the possibility of paraffinophilic organism presence. Paraffinophilic MAI organism presence can be confirmed after the assay results are known for the assays discussed hereinafter.
• Figure 4 shows the nitrate reduction assay 70.
• a slide culture 44 showing heavy growth is assayed for the ability to reduce nitrates to nitrites. This is done by adding nitrates to a tube 71 containing a sterile broth. After a period of 12 to 24 hours incubation at 37°C, the slide 44 is removed from the sterile nitrate broth 72 and five drops of sulfanilic acid reagent solution followed by five drops of alpha naphthylamine reagent solution are added to the tubes 71. The reduction of nitrate to nitrite appears as a red colored broth 73. As is known, if the nitrate is reduced to nitrite, this indicates the absence of paraffinophilic MAI organism on the slide.
• Figure 5 shows the urea hydrolysis reaction assay 80.
• a slide culture 45 is added to a plugged tube 81 containing 4.5 ml of sterile urea broth 82. The culture is incubated at 37°C and checked after a period of three days. A positive reaction involves a color change of the broth 82 to pink or red after a period of three days. As is known, if the solution changes color, this indicates the absence of a paraffinophilic MAI organism on the slide 45.
• Figure 6 shows the emulsifier hydrolysis assay 90.
• the emulsifier used is "Tween 80," a trademark of Atlas Chemical Industries, Inc. and is generically described as polyoxyethylene derivatives of fatty acid partial esters of sorbitol anhydrides.
• a slide culture 46 was added to sterile plugged tubes 91 containing media 92 and incubated at 37°C.
• a positive reaction involved the appearance of a red coloration 93 on the meniscus pellicle 94 of the slide 46 within five days. As is known, the presence of the red coloration in the slide indicates the absence of a paraffinophilic organism on the slide.
• the paraffinophilic MAI organism identification tests should be performed, with the tellurite reduction test being the most important of the four tests.
• all four of the tests should be performed in order to more accurately speciate and identify the paraffinophilic organism.
• DNA extraction portion of the process of the present invention an example will be provided.
• Figure 7 illustrates DNA extraction for ParaFecogen.
• a paraffin slide 102 with the paraffinophilic organism from tube 57 is subjected to scraping to remove the paraffin coating containing the paraffinophilic organism growth employing a flame sterilized inoculated needle 122 with the introduction of the scraped paraffinophilic organism growth being placed through funnel 126 into a microcentrifuge tube 128 containing about 1.0 ml of octane.
• the bacteria and residual paraffin are pelleted by centrifugation into a microcentrifuge operating at about 14,000 rp for about five minutes.
• This supernate (residual octane) is removed from the microcentrifuge by carefully pipetting with a sterile Pasteur pipett.
• a 0.5 ml. aliquot of 100% ethanol is added to the microcentrifuge tube 128 and the tube is mixed by gentle inversion.
• the tube is placed within a microcentrifuge and again spun down at 14,000 rpm for five minutes.
• Supernate is removed by the sterile Pasteur pipett.
• the pellet within the microcentrifuge tube 128 is once again exposed to about 0.5 ml of 100% ethanol and spun down at 14,000 rpm for five minutes. The supernate is again withdrawn with a sterile pipett.
• the residual ethanol is finally removed from the microcentrifuge tube by evaporation under a vacuum.
• An aliquot of about 0.5 ml of lysozyme (10 mg/ml) was added to the pellet contained within the microcentrifuge tubes which were then vortexed and incubated for about 20 minutes at 37°C.
• the microcentrifuge was spun down at 14,000 rpm for five minutes and the supernate was carefully removed.
• the pellet within the microcentrifuge tube was vortexed and then exposed to 0.500 ml of Proteinase K (10 mg/ml) for a period of two hours at about 50°C.
• the microcentrifuge tube was spun down in a microcentrifuge for about five minutes at 14,000 rpm.
• Patents 4,683,195; 4,683,202; 4,695,188 (the disclosures of which are incorporated herein by reference) to identify the presence of MAI sequences in the DNA pellet.
• suitable genetic amplification systems employable in the present invention is that marketed under the trade designation Polymerase Chain Reaction (PCR) by Roche Corporation.
• the invention is not so limited. In its broader aspects, the invention may be employed with other body specimens and may be employed to detect the presence of other types of paraffinophilic matter in the specimen.

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• 1994
  • 1994-01-21 JP JP6517248A patent/JPH08506483A/ja active Pending
  • 1994-01-21 WO PCT/US1994/000831 patent/WO1994017200A1/en not_active Application Discontinuation
  • 1994-01-21 CA CA 2153901 patent/CA2153901A1/en not_active Abandoned
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