AU671196B2 - Detection of paraffinophilic microorganisms - Google Patents

Description

A WO 94/17200 PCTIUS94/00831 1- Detection of Paraffinophilic Microorganisms CROSS REFERENCE TO RELATED APPLICATIONS This application is a continuation-in-part of United States Patent Application Serial No. 08/035,358, filed March 22, 1993 and entitled "A METHOD FOR PARAFFINOPHILIC ISOLATION AND IDENTIFICATION FROM A BODY SPECIMEN", which was a continuation-in-part of United States Patent Application Serial No. 08/011,479, filed on January 26, 1993 and entitled "A METHOD FOR MYCOBACTERIUM AVIUM INTRACELLULARE ISOLATION AND IDENTIFICATION FROM A FECAL SPECIMEN." BACKGROUND OF THE INVENTION 1. Field Of The Invention: This invention relates to a method for speciating and identifying paraffinophilic materials.

2. Description Of The Prior Art: 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 Mycobacterium avium intracellulare complex (MAI).

It has been found that more than 80% of AIDS patients have MAI present in their bodies Wallace et al., Mycobacterium avium complex in patients with AIDS A Clinicopathologic Study. Chest 93 926-932 (1988)). Organisms of MAI prior to the AIDS epidemic were recognized as a rare form of pneumonia in patients with N J U i 0 ~it El WO 94/17200 PCT/US94/00831 2chronic lung infections 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 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.

Studies have shown that disseminated MAI infection makes a substantial contribution to both morbidity and mortality in AIDS patients R. Horsburgh, Jr. et al., The Epidemiology of Disseminated Nontuberculous Mycobacterial Infection in the Acquired immunodeficiency syndrome (AIDS).

Am. Rev. Respir. Dis. 1989, 139:4-7).

Up to the present time, the most common source of isolating MAI clinically has been by means of the bloo.

Isolation techniques for determining the presence or absence of MAI in the patients' blood are known. One method involves using the BACTEC Radiometric System, which is a product of the Johnson Division of Becton and Dickenson. The system itself utilizes hemoculture tubes that contain Middlebrook 7H12 liquid broth plus 0.05% sodium polyanethyl sulphonate in hemoculture vials. In addition, the 7H12 broth contains Carbon-14 labelled palmitic acid. In use, vials containing mycobacterial growth give off Carbon-14 labelled CO 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 M. Reichert et al., Pathologic features of AIDS. In: V. T. DeVita Jr. et al. j (eds) AIDS etiology, diagnosis, "reatment and prevention, p. 134 NY J. 13, Lipponcott, 1985).

1 -1 i )i r r r i j WO 94/17200 PCT/US94/00831 3 However, when MAI becomes widely disseminated in AIDS patients the involvement by way of the blood of bone, lungs, spleen and the CNS causes an almost 100% rate of mortality C. Hawkins et al., MAI in patients with acquired immunodeficiency syndrome. Am. Intern. Med. 1986; 105: 184-8) Hoy et al., Quadruple drug therapy for Mycobacterium avium intracellulare bacteremia in AIDS patients. J. Infect. Dis.

990; 161: 801-5).

These known methods, although effective, require expensive equipment and specialized operating personnel and materials. Thus, smaller hospital centers where few AIDS patients are seen, field laboratories, and third world countries, where resources are limited, do not have this specialized equipment and personnel. A simpler and more inexpensive method and apparatus of isolating and identifying MAI would be of substantial benefit in such situations.

It is known that many atypical Mycobacteria grow on basal salt media devoid of any carbon sources other than paraffin wax which is introduced into the media in the form of paraffin was coated roads. Fuhs, G. "Der Mikrobiell Abbau Von Kohlenwasserstoffen", Arch. Mikrobiol. 39:374-422 (1961).

Mishra, S. K. et al., "Observations On Paraffin Baiting As a Laboratory Diagnostic Procedure in Nocardiosis", Mycopathologica and Mycologia Applicata 51 147-157 (1973) utilized paraffin coated rods and basal salt medium to isolate Nocardia asteroides from clinical specimens such as sputum, bronchial lavage and cerebrospinal fluid.

The technique was further improved by substituting paraffin wax coated slides for rods and thereby making possible the use on an in situ Kinyoun cold acid-fastness staining procedure for organisms growing on the paraffin coated slide. Ollar, R. "A Paraffin Baiting Technique that Enables a Direct Microscopic View of in situ Morphology of Nocardia asteroides with the Acid-Fast or Fluorescence X.1 1 k jj i' f 1 i- WO 94/17200 PCT/US94/00831 4 Staining Procedures", Zbl. Bakt. Hyg., Abt. Orig. A, 234: 81-90 (1976). With this assay, a positive reaction tells the user immediately that a mycobacteria organism other than M. tuberculosis is present.

United States Patent No. 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.

As will be apparent from the foregoing, it is vital to human health that MAI be identified and treated as early as possible A. Kemper'et al., California Collaborative Group; Microbiologic and clinical response of patients with AIDS and MAC bacteremia to a four oral drug regimen; In: Program and abstracts of the 30th Interscience Conference on Antimicrobial Agents and Chemotherapy; Atlanta, October 21-24, 1990; Washington Am. Society for Microbiology; 1990; 297.

abstract).

There is a real and substantial need for improved means of rapidly determining the presence of MAI and other paraffinophilic organisms in a patient.

SUMMARY 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 nay be bacterial or fungal in nature.

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i i pCTIJS94/00 8 31 WO 94/17200 5 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. If the result of this test determines that an alcohol-acid fast organism is present, 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.

is an object of the present invention to isolate, identify and speciate paraffinophilic organisms in a body specimen in a rapid and reliable manner.

It is a further object of the invention to provide such a system which employs DNA extraction as a means for expediting prompt results.

It is a further object of the invention to provide a paraffinophilic organism specific simplified and rapid system which effects isolation and speciation of a 30 paraffinophilic organism from fecal matter or other body specimens.

~ii j, r WO 94/17200 PCTI/US94/00831 -6- It is a further object of the present invention to employ the system of this invention in ascertaining whether a particular human has a paraffinophilic organism present.

It is a further object of the invention to provide a non-invasive method of detecting the presence of a paraffinophilic organism in a patient.

These and other objects of the invention can be more fully understood with reference to description and the drawings appended to this application. i BRIEF DESCRIPTION OF THE DRAWINGS 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 acidalcohol fastness assay employable in the present invention. I 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.

Figure 6 is a schematic illustration of a Tween hydrolysis.

Figure 7 is a schematic illustration of the use of DNA extraction in accordance with the present invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS As used herein, 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.

u A WO 94/17200 PCTIUS94/00831 7 As used herein, the term "body specimen" shall include fecal matter, blood, sputum, tissue, and cerebral spinal fluid obtained from a patient.

As used herein, the term "paraffinophilic" shall expressly include, but not be limited to the following organisms: Micrococcus Paraffinae; Corynebacterium Simplex; Ahnl; Mycococcus (Rhodococcus) Cinnabareus; Ahnl. Mvcococcus (Rhodoc) Rhodochrous; Mycobact. Perrugosum Var. Athanicum; Mycobact. Rubrum Var. Propanicum; Mycobacterium Hyalinum; Mycobacterium Lacticola; Mycobacterium Album, M. Luteum; Mycobacterium Microti; Mvcobacterium Rubrum. Mvcobacterium Phlei,; Mycobacterium Phlei. M. Smeqmatis; Mycobacterium Testudo; Mycobacterium-Avium-Intracellulare; Nocardia Spp.; Actinomyces; Candida Lipolytica; Candida Tropicalis, Torulopsis Colliculosa; Monilia Sp., Hansenula Sp., Torula rossa; Penicillium Sp.; IHNL. Asperqillus Flavus; Asperqillus sp., Penicillium Sp.; Citromyces Sp..

Scopulariopsis Sp.; Pseudomonas Fluorescens Liquefaciens; Ahnl, Pem. Fluorescens Denitrificans; Pseudomonas Aeruqinosa.

In addition to the MAI detection in fecal matter, it is important to be able to detect other paraffinophilic organisms in other body specimens in order to facilitate rapid and accurate diagnostic determination of conditions having significant impact on a patient's health. 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. Such conditions can lead to undesired opportunistic infections, the early detection of which can be of great importance to proper treatment of a patient. Among the most important categories of such paraffinophilic organisms are the mycobacterium, candida and nocardia species.

J i i WO 94/17200 PCTIUS94/00831 8 As indicated hereinbefore, it is vital to human health that MAI be identified and treated as early as possible A. Kemper et al., California Collaborative Group; Microbiologic and clinical response of patients with AIDS and MAC bacteremia to a four oral drug regimen; In: Program and abstracts of the 30th Interscience Conference on Antimicrobial Agents and Chemnotherapy; Atlanta, October 21-24, 1990; Washington Am. Society for Microbiology; 1990; 297.

abstract).

The identification of MAI in fecal matter is advantageous in accomplishing this objective as MAI is initially found in the gastrointestinal tract 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 submucosal layers of the gastrointestinal tract.

It will be appreciated that early detection of MAI can result in significant prolongation of the life of an AIDS patient.

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 E. Bermudez et al., An animal model of Mycobacterium avium complex disseminated infection after colonization of the intestinal tract; Kuzell

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*L t WO 94/17200 PCT/US94/00831 9- Institute for Arthritis and Infectious Diseases; Medical Research Institute of San Francisco at California Pacific Medical Center 94115; J. -nfect. 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 A. Murphy et al., Mycobacterium avium intracellulare in a hospital hot water system: epidemiological investigation; In: Proceedings and abstracts of the 24th Interscience Conference on Antimicrobal 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, Ma et al., AIDS and Infections of Homosexual Men Second Edition, Appendix 14-B; Stoneham, MA; Butterworths, 1989; 233-234) may be in large part a manifestation of MAI infection. The shortened survival time (median longevity: 4.1 months with disseminated MAI infection) A. Havlik, Jr.

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L i WO 94/17200 PCT/US94/00831 10 et al., Disseminated Mycobacterium avium complex infection: clinical identification and epidemiologic trends; J. Infect.

Dis. 1992; 165: 577-80) is presumed due to the severe loss of weight. In many cases, death has been found to result from inanition. It would, therefore, be advantageous to have monthly checks for MAI in all patients who meet the following reasonable criteria: 1) HIV positivity, 2) loss of weight greater than 5 lbs. or greater than 5% of body weight, and 3) a CD4 cell count under 400. If MAI is detected in the HIV patient's stool using the above criteria, the use of eR.13y aggressive antibiotic therapy has been shown to prevent dissemination. Recent therapy has involved the use of Ciprofloxacin with Ethambutol, Ansamycin and Clofazimine B. Inderlied et al., Disseminated mycobacteriur, avium complex infection; AIDS Clin. Rev. 1990; 165-91). However, each individual HIV patient's antibiotic regimen would be guided by the use of antibiotic sensitivity testing such as that done by the MAI Para SL/C (Paraffin Slide Culture) method.

In the past, detection of these silent MAI infections of the G.I. tract in the ARC patient was very time consuming when the Lowenstein-Jensen, Middlebrook 7H9 and related Middlebrook media were used. Very lengthy protocols involving centrifugation, isolation and slow growth made these prior methods very impractical for use in the periodic testing of the G.I. tract in HIV patients for MAI. A simplified and inexpensive method specifically for handling contaminated fecal sample is needed for such periodic screening of HIV patients for MAI. 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

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h" Is WO 94/17200 PCT/US94/00831 11 i presence of gastrointestinal in accordance with the above criteria.

The initial portion of the method of the present invention is the specific embodiment of that disclosed in Ollar, United States Patent No. 5,153,119. In isolating the paraffinophilic organism from the body specimens, a suspension may be prepared by suspending a 4mm loopful of fresh body specimen in 3 to 5 milliliters of sterile saline. Employing 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. 5,153,112° 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 contamjiating 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 7 other hand because the latter cannot utilize paraffin was as a sole source of carbon. As is known, 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 1 0 WO 94/17200 PCT/US94/00831 12 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 (Figure This test can be used to further distinguish between the atypical mycobacteria and the nocardioform organisms. As is known, atypical paraffinophilic mycobacteria are alcohol-acid fast; paraffinophilic nocardioform organisms are acid-fast and paraffinophilic Pseudomonas aeruqinosa or paraffinophilic Candida tropicalis are neither acid nor alcohol-acid fast. Thus, these latter two paraffinophilic groups (nocardioforms and Pseudomonas aeruainosa or Candida tropicalis) can be eliminated as possibilities by the alcoholacid'fastness testing.

In isolating the paraffinophilic organisms as shown in Figure 2, the eight test tubes 50, 51, 52, 53, 54, 56, 57, are each provided with about 4.5 milliliters of sterile Czapek broth containing the antibacterial and antifungal/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 0 C. When growth is observed, typically after about 24 P? to 96 hours, 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 WO 94/17200 PCTIUS94/00831 13 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 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 1 tube 12 of Figure 1 and is first immersed in two consecutive i 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 HCl for five minutes.

After this, 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. Finally, 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 microscore at 250x, 450x and 1000x 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 incubateC. 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 VO 94/17200 PCT/US94/00831 14 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 0 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 0 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 Figure 6 shows the emulsifier hydrolysis assay 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 0

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.

It will be appreciated that at least the first three of the paraffinophilic MAI organism identification tests tellurite reduction, nitrate reduction assay, urea hydrolysis assay, or "Tween 80," or hydrolysis emulsifier assay) should be performed, with the tellurite i'rj i I

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r r i 1 i, i i: ii 1 1 t i rj E i i! 7 WO 94/17200 PCT/US94/00831 reduction test being the most important of the four tests.

Preferably, all four of the tests should be performed in order to more accurately speciate and identify the paraffinophilic organism.

With the completion of these sequences, the basic speciation and isolation of paraffinophilic organism for a body specimen sample is completed. The final processing preferably involves DNA extraction.

Example 1 In order to provide further guidance regarding the DNA extraction portion of the process of the present invention, an example will be provided.

Figure 7 illustrates DNA extraction for ParaFecogen.

With reference to Figure 7, 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 ml of octane. The bacteria and residual paraffin are pelleted by centrifugation into a microcentrifuge operating at about 14,000 rpm 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 WO 94/17200 PCTIJS94/00831 -16 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 0 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 mg/ml) for a period of two hours at about 50 0 C. The microcentrifuge tube was spun down in a microcentrifuge for about five minutes at 14,000 rpm. The supernate was then carefully removed. An aliquot of 100 ul of isopropanol was then added to the pellet contained within the microcentrifuge tube and the tubes were spun at 14,000 rpm for 30 minutes at 4 0 C. The supernate was removed and the pellet was exposed to 100.ul of 70% ethanol, placed in a microcentrifuge and spun down at 14,000 rpm for 15 minutes. The supernate was removed and the DNA containing pellet was resuspended in ul TE buffer (10 mM Tris-HCl, 1mM EDTA, pH The resulting DNA can then be utilized with other DNA methodologies such as dot blot, southern blot, or gene amplification, such as is di-...osed in U.S. 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. Among the suitable genetic amplification systems employable in the present invention is that marketed under the trade designation Polymerase Chain Reaction (PCR) by Roche Corporation.

It will be appreciated that the foregoing system rz provides an improved means for rapidly and effectively V 30 isolating and identifying paraffinophilic MAI organisms present in a body specimen.

It will be appreciated that while a substantial portion of the disclosure has focused on the isolating and identifying of MAI, with particular emphasis on employing S 'i WO 94/17200 PCT/US94/00831 i -17 fecal matter as the body specimen, 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.

Whereas particular embodiments of the invention have been described herein for purposes of illustration, it will be evident to those skilled in the art that numerous variations i of the details may be made without departing from the invention as defined in the appended claims.

Claims (11)

1. A method of determining the presence of a paraffinophilic organism in a body specimen comprising: introducing portions of said body specimen into a plurality of receptacles containing a sterile broth and antibiotics; introducing one paraffin coated slide into each said receptacle; observing said paraffin coated slide for the presence of organisms growing thereon; after observing organism growth on said paraffin coated slides, performing an alcohol-acid fastness test on a first said paraffin coated slide to determine whether said organism is an alcohol-acid fast organism, an acid-fast organism or a non-acid fast/non-alcohol-fast organism; after performing said alcohol-acid fastness test on said first paraffin coated slide and determining that an alcohol-acid fast organism is present on said first paraffin coated slide, performing a tellurite reduction assay on a second said paraffin coated slide to determine the possibility of the presence of a paraffinophilic organism on said second paraffin coated slide; after performing said Stellurite reduction assay on said second paraffin coated slide and determining that there is S 15 a possibility of the presence of a paraffinophilic organism on said second paraffin coated slide, performing at least one speciation assay on a third said paraffin coated slide to confirm the presence of a paraffinophilic organism on said third paraffin coated slide; subsequently performing a DNA extraction on at least one additional said slide; and o determining from said DNA extraction slide whether a paraffinophilic organism is present in said specimen.

2. The method of claim I including performing as said speciation assay a nitrate reduction assay. b t. e

3. The method of claim 2 includingA to said nitrate reduction assay performing on a fourth said paraffin coated slide a urea hydrolysis reaction assay.

4. The method of claim 3 including to said urea hydrolysis reaction assay performing on a fifth said paraffin coated slide an emulsifier hydrolysis assay.

The method of claim 1 including employing said method on at least one body specimen selected from the group consisting of fecal matter, blood, sputum, tissue, and cerebral spinal fluid. po O 2 30

6. The method of claim 5 including( said method to test a fecal specimen of a human being to determine if said human being has MAI or other paraffinophilic organism.

7. The method of claim 1 including/ nl said method to test for 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- 4Jvium-Intracellulare; Nocardia Spp.; Actinomyces; Candida Lipolytica; Candida I -I r 4 6 i .v In:\libcl01046:JOC i i; m 1 p.- 2 r Tropicalis, Torulopsis Colliculosa, Monilia Sp., Hansenula Sp., Torula rossa; Penicillium Sp.; IHNL. Aspergillus Flavus; Aspergillus sp., Penicillium Sp.; Cit romyces Sp., Scopulariopsis Sp.; Pseudomonas Fluorescens Liquefaciens; Ahnl, Pem. Fluorescens Denitrificans; Pseudomonas Aeruginosa. s

8. The method of claim 7 including employing said method on a human body s p e c i m e n ,s m t o n a h m r e a

9. The method of claim 8 including,( 's1~mto o ua ea specimen to determine if said human has said paraffinophilic organism.

The method of claim 9 including employing said method to determine if said human has MAI.

11. A method of determining the presence of a paraffinophilic organism in a body specimen, substantially as hereinbefore described with reference to any one of the Example. Dated 21 July, 1995 is Infectech Inc. I ft o 9 5~999 A 9 *94 99 9 e9 9. 9 I I II ''It Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON 9,4 4 I 094 9 949I 9, .9 4 .4 .9 994 44 99 4 9 5J 9 o '9 99 [n:\Iibc1 046:JOC