Note: Descriptions are shown in the official language in which they were submitted.
WO94/17200 2 15 ~ 90 1 PCT~S94/00~1
-- 1 --
Det~;lion 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, l9g3 and entitled
"A METHOD FOR MYCOBACTERIUM AVIUM INTRACELLULARE ISOLATION AND
IDENTIFICATION FROM A FECAL SPECIMEN."
10~ACKGROUND OF THE INVENTION
1. Field Of The Invention:
This invention relates to a method for speciating
and identifying paraffinophilic materials.
2. Descri~tion Of The Prior Art:
15Human 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 (J.M. Wallace
et al., Mycobacterium avium complex in patients with
AIDS - A Clinicopathologic Study. Chest 93 (5) 926-932
(1988)). Organisms of MAI prior to the AIDS epidemic were
recognized as a rare form of pneumonia in patients with
WO94/17200 PCT~S94100~1
21~3901 - 2 -
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.
Studies have shown that disseminated MAI infection
makes a substantial contribution to both morbidity and
mortality in AIDS patients (C. 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 blood.
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% (v/v) 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 CO2 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).
WO94/17200 215 3 9 0 1 PCT~S94/00~1
-- 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. C. Hawkins et al., MAI in patients with acquired
immunodeficiency syndrome. Am. Intern. Med. 1986; 105: 184-8)
(J. 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. W., "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 (2-3): 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., "A Paraffin Baiting Technique
that Enables a Direct Microscopic View of in situ Morphology
of Nocardia asteroides with the Acid-Fast or Fluorescence
WO94/17200 PCT~S94/00~1
21539~1
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
~uite 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 (C. 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 D.C.; 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 INVENTI~)N
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 may be bacterial or fungal in nature.
WO94/17200 PCT~S94/00~1
~1~3~01 ~
-- 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.
It 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
paraffinophilic organism from fecal matter or other body
specimens.
.
WO94/17200 ~15 3 9 ~1 PCT~S94/00~1
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.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure l 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.
Figure 6 is a schematic illustration of a Tween 80
hydrolysis.
Figure 7 is a schematic illustration of the use of5 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.
WO94/17200 PCT~S94/00~1
2153~01
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. 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; 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.
~ 2~i39~, --
WO94/17200 r PCT~S94/00~1
As indicated hereinbefore, it is vital to human
health that MAI be identified and treated as early as possible
(C. 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 D.C.; 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 (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
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 (L. E. Bermudez et al.,
An animal model of Mycobacterium avium complex disseminated
infection after colonization of the intestinal tract; Kuzell
WO94/17200 21 5 3 9 0 I PCT~S94/00~1
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 the24th 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
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) (J. A. Havlik, Jr.
WO94117200 21 S 3 9 01 PCT~S94/00~1
-- 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 early
aggressive antibiotic therapy has been shown to prevent
dissemination. Recent therapy has involved the use of
Ciprofloxacin with Ethambutol, Ansamycin and Clofazimine
(C. B. Inderlied et al., Disseminated mycobacterium 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 ~uick and inexpensive means of early detection of the
W094/17200 PCT~S94/00~1
~1 53~01
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 subse~uently 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 aeruginosa 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. 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
WO94/17200 2 ~ 5 3 9 ~1 PCT~S94/00~1
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. As is known, 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. Thus, these latter two paraffinophilic
groups (nocardioforms and Pseudomonas aeruqinosa or Candida
tropicalis) can be eliminated as possibilities by the alcohol-
acid fastness testing.
In isolating the paraffinophilic organisms as shown
in Figure 2, the eight 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 37C. When growth is observed, typically after about 24
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
WO94/17200 i~'; f i PCT~S94/00~1
;2~39~1
- 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 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 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 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
WO94/17200 PCT~S94100~1 ~
2~L53901
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 37C, 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 37C 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 9O.
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 37C.
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
((1) tellurite reduction, (2) nitrate reduction assay,
(3) urea hydrolysis assay, or (4) "Tween 80," or hydrolysis
emulsifier assay) should be performed, with the tellurite
W094/17200 21~ 3 ~ 0 1 PCT~S94/00~1
- 15 -
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
1.0 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
W094/17200 - PCT~S94/00~1
~ 215t39~3l
- 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 37C. 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 50C. 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
4C. 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
50 ul TE buffer (10 mM Tris-HCl, lmM EDTA, pH 8.0). The
resulting DNA can then be utilized with other DNA
methodologies such as dot blot, southern blot, or gene
amplification, such as is disclosed 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
provides an improved means for rapidly and effectively
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
WO94/17200 21~ 3 9 01 PCT~S94/00~1
, j . ~
- 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
of the details may be made without departing from the
invention as defined in the appended claims.
