U.S. patent application number 13/773874 was filed with the patent office on 2013-06-27 for system for detecting infectious agents using computer-controlled automated image analysis.
This patent application is currently assigned to Ikonisys, Inc.. The applicant listed for this patent is Michael Kilpatrick, Triantafyllos P. Tafas, Petros Tsipouras. Invention is credited to Michael Kilpatrick, Triantafyllos P. Tafas, Petros Tsipouras.
Application Number | 20130163845 13/773874 |
Document ID | / |
Family ID | 23138345 |
Filed Date | 2013-06-27 |
United States Patent
Application |
20130163845 |
Kind Code |
A1 |
Tafas; Triantafyllos P. ; et
al. |
June 27, 2013 |
System For Detecting Infectious Agents Using Computer-Controlled
Automated Image Analysis
Abstract
A method for providing quantitative information regarding the
extent of infection of host cells by an infectious agent. A
microscope image of a specimen of a bodily fluid is analyzed using
image processing techniques to quantify the percentage of the area
of the specimen that is infected.
Inventors: |
Tafas; Triantafyllos P.;
(Rocky Hill, CT) ; Kilpatrick; Michael; (West
Hartford, CT) ; Tsipouras; Petros; (Madison,
CT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Tafas; Triantafyllos P.
Kilpatrick; Michael
Tsipouras; Petros |
Rocky Hill
West Hartford
Madison |
CT
CT
CT |
US
US
US |
|
|
Assignee: |
Ikonisys, Inc.
New Haven
CT
|
Family ID: |
23138345 |
Appl. No.: |
13/773874 |
Filed: |
February 22, 2013 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
13188714 |
Jul 22, 2011 |
8385625 |
|
|
13773874 |
|
|
|
|
13022348 |
Feb 7, 2011 |
7991213 |
|
|
13188714 |
|
|
|
|
12052898 |
Mar 21, 2008 |
7885449 |
|
|
13022348 |
|
|
|
|
10543270 |
Jun 2, 2006 |
|
|
|
PCT/US02/17586 |
Jun 4, 2002 |
|
|
|
12052898 |
|
|
|
|
60295587 |
Jun 4, 2001 |
|
|
|
Current U.S.
Class: |
382/133 |
Current CPC
Class: |
C12Q 1/04 20130101; G01N
15/1475 20130101; G06T 7/0012 20130101; G06K 9/00127 20130101; G06T
2207/30024 20130101; G06K 9/00147 20130101 |
Class at
Publication: |
382/133 |
International
Class: |
G06K 9/00 20060101
G06K009/00 |
Claims
1. A computer software product, comprising a computer-readable
storage medium having fixed therein a sequence of instructions
which, when executed by a computer, direct the performance of a
method which comprises: a) acquiring a microscope image of an
optical field of a substrate having fixed thereon a monolayer of
animal cells which either produce or are treated to produce a first
signal specific to an animal cell of interest and treated to
produce a second signal specific to an infectious agent of
interest, if present, and transferring the image to an ROB image;
b) transferring the Red component of the RGB image to a new
monochrome grey image; c) transforming the grey level image to a
binary image using a cut off point set to a value indicative of the
expected size of the animal cells of interest; d) operating on the
binary image to remove noise and fill holes; e) measuring size of
the image of step d), selecting and recording areas representative
of the animal cells of interest; f) transferring the Red component
of the original ROB image to a second binary image using an
expected value indicative of the infectious agent of interest; g)
transferring the Green component of the original RGB image to grey
level; h) forming a new grey level image using all pixels having a
value equal to a set value M and any grey level value in the Green
component in the original RGB image equal to a set value N; i)
transforming the new grey level images to a third binary image; j)
operating on the third binary image to remove noise and fill holes;
k) recording the area of the third binary image after step j); and
l) determining whether or not, each identified animal cell is
occupied by an infectious agent of interest.
2. The computer software product in accordance with claim 1,
wherein the method further comprises calculating the extent of each
animal cell occupied by said infectious agent.
3. The computer software product in accordance with claim 1,
further comprising a computer-readable storage medium having fixed
therein a sequence of instructions which, when executed by a
computer, direct the performance of a method which comprises: a)
acquiring a microscope image of an optical field of a substrate
having fixed thereon a sample of a body fluid or tissue from an
animal patient suspected of containing an infectious agent of
interest, which sample either produces or is treated to produce a
first signal specific to an infectious agent of interest, if
present, and transferring the image to an RGB image; b)
transferring the RGB image to an HLS image and transforming the HIS
image to a new monochrome grey level image; c) transforming the
grey level image to a binary image; d) operating on the binary
image using at least one filter to identify blobs; and e) selecting
blobs based on the expected size of area of an infectious agent to
determining whether or not an infectious agent of interest is
present in the sample.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This is a continuation application of U.S. patent
application Ser. No. 13/188,714, filed Jul. 22, 2011, which is a
continuation application of U.S. patent application Ser. No.
13/022,348, filed Feb. 7, 2011, which is a continuation of U.S.
patent application Ser. No. 12/052,898 filed Mar. 21, 2008, now
U.S. Pat. No. 7,885,449, which is a continuation of U.S. patent
application Ser. No. 10/543,270, filed Jun. 2, 2006, which is a
National Stage of International PCT Application Number
PCT/US02/17586 filed on Jun. 4, 2002 and published as WO02/098280
on Dec. 12, 2002, which claims priority to U.S. Provisional Patent
Application No. 60/295,587 filed Jun. 4, 2001, each of which is
incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to computer controlled methods
and apparatus for detecting or detecting and quantifying an
infectious agent in a biological sample. The identifying and/or
quantitative data obtained are useful in making a diagnosis or
prognosis of diseases, including diseases classically deemed
"non-infectious." in one aspect, the invention relates to computer
controlled methods and systems for identifying an infected host,
animal or plant cell in a field of cells and thereby providing
information useful in making a disease diagnosis or a prognosis of
disease susceptibility based on identification of an infected
animal cell. In another aspect, the invention relates to computer
controlled methods and systems for quantification of infected host,
e.g., animal or plant cell(s) in a biological sample, thereby
providing information useful in making a disease diagnosis or a
prognosis of disease susceptibility based on the quantification of
infected cell(s). The quantification may be determination of the
number of infected animal cells, or of the extent to which the
cells are infected (i.e., the number of infectious agents in an
infected cell) or determination of both numbers. The quantitative
information also is useful to assess therapeutic efficacy of
treatment of disease.
[0003] In one important embodiment, the invention relates to
quantification of Chlamydia infected mononuclear phagocyte(s) in a
blood or tissue sample to provide information useful to make a
diagnosis or prognosis of susceptibility of vascular or coronary
disease.
[0004] In another important embodiment, the invention relates to
quantification of Chlamydia infected mononuclear phagocyte(s) in a
biological sample to provide information helpful for or useful to
make a diagnosis (or prognosis) of a central nervous system (CNS)
disease or disorder.
[0005] In yet another aspect, the present invention relates to
computer controlled methods and systems for detecting and
quantifying an infectious agent "free floating" in a biological
sample to provide data useful in making a diagnosis or prognosis of
disease or disorders, including diseases classically deemed
"non-infectious".
BACKGROUND OF THE INVENTION
[0006] Citation or identification of any reference in this section
or any section of this application shall not be construed as an
admission that such reference is available as prior art to the
present invention.
[0007] Atherosclerosis is the main underlying cause of coronary
heart disease and is characterized by the deposit of lipid
containing plaques on endothelium of large and medium sized
arteries. Atherosclerosis is thought to be initiated at
dysfunctional vascular endothelium when normal laminar blood flow
is disrupted. Many systemic and local factors may cause
dysfunctional endothelium and lead to or trigger an inflammatory
response in the vessel wall. Multiple cell types can mediate this
process, including monocyte-derived macrophages. Drexler, H. 1997.
Prog. Cardiovasc. Dis. 39:287. The dysfunctional endothelium allows
passage of low density lipoprotein ("LDL") cholesterol and
expresses multiple adhesion molecules for platelets and
inflammatory cells. The LDL cholesterol undergoes partial oxidation
and causes further endothelial dysfunction while monocytes
penetrate the endothelium, differentiate into macrophages, and take
up oxidized LDL cholesterol. The resulting lipid-laden macrophages,
also known as foam cells, accumulate in the atherosclerotic lesion
and ultimately may rupture to release oxidized LDL cholesterol and
cytotoxic enzymes. This triggers fibroproliferative responses from
vascular smooth muscle cells and leads to the development of
atherosclerotic plaques. Fuster et al. 1992. N. Engl. J. Med.
326:310; Stary, H. C. 1989. Arteriosclerosis 99:1-19.
[0008] Studies have shown Herpes viruses, such as Herpes simplex
virus and Cytomegalovirus ("CMV"), can increase the risk of
developing heart disease. Roivainen et al. 2000, Circulation
101(3):252. For example, elevated CMV antibody titres are
associated with the presence of atherosclerosis. Melnick et al.
1990. JAMA 263:2204; Danesh et al. 1997, Lancet 350:430; Cheng et
al. 2000, Expert Opin. Investig. Drugs 9(11):2505. Based on
pathological data demonstrating CMV DNA sequences and viral
inclusions in atherosclerotic lesions, 75 consecutive patients
undergoing directional coronary atherectomy for coronary disease
were studied to see if a link between CMV infection and arterial
disease exists. The results showed that patients who were
seropositive for CMV prior to the procedure have a greater than
five-fold increased rate arterial disease. Zhou et al. 1996. N.
Engl. J. Med. 335:624.
[0009] A mechanism by which CMV may affect atherosclerosis hinges
on the mononuclear phagocyte. CMV integrates into mononuclear cell
precursor DNA thereby causing circulating monocytes to be a vector
for delivering virus to sites of vessel inflammation. Guetta et al.
1997. Circ. Res. 81:8. Macrophages have been shown to be a similar
source of circulating HUV in patients with AIDS. Orenstein et al.
1997. Science 276:1857. Studies have demonstrated that endothelial
cells, smooth muscle cells, and oxidized LDL cholesterol can
activate CMV viral replication in infected mononuclear phagocytes
which can lead to macrophage, endothelial cell, and vascular smooth
muscle cell infection with CMV. CMV infected smooth muscle cells
may then obtain growth advantages and contribute to proliferative
responses in atherosclerosis due to CMV induced changes in
expression of regulatory proteins. Speir et al. 1994. Science.
265:391.
[0010] A link between atherosclerosis and Helicobacter pylori has
also been shown. H. pylori is a Gram-negative rod which has been
implicated in the development of peptic ulcers, gastric carcinoma,
and low-grade B cell lymphomas of the gastrointestinal tract.
Schussheim et al. 1999, Drugs 57:283. An association of H. pylori
infection with coronary disease has been suggested in which
seropositivity conferred a two-fold increased risk of coronary
artery disease among nearly 200 men. Mendall et al. 1994. Br. Heart
J. 71:437; Danesh et al. 1997, Lancet 350:430; Cheng et al. 2000,
Expert Opin. Investig. Drugs 9(11):2505; Muhlestein, J. B. 2000,
Curr. Interv. Cardiol. Rep. 2(4):342. Another study supports this
association when it was seen that elevated serum fibrinogen levels
and total leucocyte count were found more often in those
seropositive for H. pylori. Patel et al. 1995. B.M.J. 311:711.
[0011] A link between Chlamydia pneumoniae and vascular disease,
such as atherosclerosis and coronary disease or coronary syndrome
is also recognized. Schussheim et al., 1999, Drugs 57:283;
Roivainen et al. 2000, Circulation 101(3):252; Muhlestein Curr.
Interv. Cardiol. Rep. 2(4):342; Danesh et al. 1997, Lancet 350:430;
Cheng et al. 2000, Expert Opin. Investig. Drugs 9(11):2505;
Muhlestein, J. B. 2000, Curr. Interv. Cardiol. Rep. 2(4):342.
Although Chlamydia is able to infect a number of cell types, the
bacteria's ability to infect mononuclear phagocytes is thought to
be pivotal to its role in the development or modulation of vascular
disease, especially atherosclerosis. Mononuclear phagocytes are
thought to spread infection from the respiratory tract to other
organ systems based upon Chlamydia to remain metabolically active
for at least 10 days in mononuclear phagocytes infected in vitro.
Moazed et al. 1998. J. Infect Des. 177:1322. Chlamydia can also
stimulate the secretion of proinflammatory cytokines such as tumor
necrosis factor-.alpha., interleukin [IL]-1 and interferon-.gamma.
from monocytes and T cells. Saikku, P. 1997. J. Infect. Dis.
104:53; Kol et al. 1998. Circulation 98:300; Hahne, S. 1997. Scand.
J. Immunol. 45:378.
[0012] Several seroepidemiological studies now associate Chlamydia
infection with atherosclerosis and promote the organism as a major
pathological factor of this general disease process. Saikku, P.
1997. Scand. J. Infect. Dis. 104:53; Campbell et al. 1998. Emerg.
Infect. Dis. 4:571. Recently, C. pneumoniae-reactive T lymphocytes
have been detected in human atherosclerotic plaques of the carotid
artery. Mosorin, M., 2000, Arterioscler. Thromb. Vase. Biol.
20:1061. The authors of this study suggest that Chlamydia, which is
commonly detected in atherosclerotic plaque of the carotid and
coronary arteries, causes T-cell activation and accumulation and
this contributes to the maintenance of the inflammatory reaction in
artherogenesis.
[0013] At least one epidemiological study has found that Chlamydia
may be present as an associated agent in neurological infections.
See, Koskiniemi, M. et al., 1996, Europ. Neurol., 36(3):60-63. A
human.
SUMMARY
[0014] In its most general aspect, the present invention provides a
computer-implemented method of detecting at least one signal, which
provides information which has diagnostic or prognostic
significance.
[0015] In its most general embodiment, the method of the present
invention includes acquiring image data of a sample of cells or a
body fluid, processing the image data to select and record images
of a detectable signal indicative of an infectious agent. Counts
may be maintained of the number and/or strength of the detectable
signal identified. The infectious agent may be contained within the
image of an animal cell or free floating in the body fluid. Such
counts provide for calculation of the extent of infection with an
infectious agent(s).
[0016] In an embodiment, the image data is transformed from one
color space, RGB (Red Green Blue) image into another color space,
e.g., HLS (Hue Luminescence Saturation) image. Filters and/or masks
are utilized to distinguish those cells that meet pre-selected
criteria, i.e. contain a detectable signal, and eliminate those
that do not, and thus identify infected animal cells.
[0017] According to one embodiment, an infectious agent is detected
or preferably detected and quantified by computer controlled image
analysis of host cells, e.g., animal or plant cells. In a preferred
embodiment, the host cells are animal cells in a sample of a body
fluid or tissue from an animal. Using this preferred method, animal
cells infected with an infectious agent are detected or preferably
detected and quantified. In the discussion below, animal cells are
discussed as an exemplary "host" cell. As would be understood by
those skilled in the art, this is for discussion purposes only and
the method would be understood to be useful, in context, for
detection of an infectious agent in any "host", i.e., animal or
plant cell.
[0018] According to this preferred embodiment of the method of the
invention, computer controlled image analysis is conducted on a
sample of body fluid or tissue containing animal cells in a
monolayer treated to provide at least two different signals which
can both be detected and quantified. At least two signals are
required. A first signal is employed to identify an animal cell of
interest and a second signal is employed to identify an infectious
agent within an identified animal cell of interest. Detection and
quantification of the two different signals provides for
determination of: (1) the number of infected animal cells, e.g.,
per unit volume of body fluid, (2) the number of infectious agents
per animal cell or (3) both the number of infected animal cells and
the number of infectious agents per cell, i.e., the extent of cell
infection.
[0019] According to the method, a monolayer of animal cells, fixed
to a suitable solid substrate is observed by a computer controlled
microscope system as described above. The monolayer can be obtained
merely by spreading a body fluid or tissue sample with animal cells
of interest on a solid substrate, such as a slide. Alternatively, a
monolayer can be obtained by spreading a sample containing an
enriched population of animal cells of interest on a solid
substrate.
[0020] A physical feature of the animal cells can be used to
provide a first signal, or more preferably the animal cells are
stained to produce a first signal. The fixed animal cells are also
treated to produce a second signal specific to an infectious agent,
if said agent is present.
[0021] The computer controlled image analysis of this embodiment of
the invention is accomplished using a computer software product
including a computer-readable storage medium having fixed therein a
sequence of instructions which, when executed by a computer directs
the performance of method steps comprising:
[0022] A microscope image of an optical field of a substrate having
fixed thereon a monolayer of animal cells treated to produce a
first signal specific to a desired animal cell and a second signal
specific to a desired infectious agent is acquired and transferred
to the computer as an RGB image.
[0023] The Red component of the RGB image is transferred to a new
monochrome grey-level image and clipped for pixel values of less
than 50 to cut down signal noise.
[0024] The grey-level image is transformed to a binary image, a
black and white image in which pixels with corresponding pixels in
the Luminance image having grey-level values lower than the cut off
point are set to a value indicative of the expected size of animal
cells of interest (white).
[0025] An opening filter, successive applications of an erosion
filter followed by a dilation filter, is applied for the removal of
small noise particles from the binary image.
[0026] Application of a hole filling function fills the holes in
the identified images.
[0027] The area of each image is measured and all images having an
area of pixels equal to or greater than the expected value of the
animal cell of interest are selected as representative of animal
cells. Cell images that have an area less than said pixels are
excluded from further processing.
[0028] The area, in pixels, of the cell images, i.e., animal cells,
is recorded and saved for farther processing. In one embodiment, in
which all cell images are to be quantified in a sample, the XY
location of each cell image is recorded.
[0029] The Red component from the original RGB image is transferred
to a binary image, so that pixels having grey-level values less
than the expected value of the signal indicative of an infectious
agent are set to 0 while all the rest are set to 255.
[0030] The Green component from the original RGB image is
transferred to grey level image.
[0031] All pixels that have a value equal to a set value M and any
grey-level value in the Green component of the original RGB image
equal to a set value N, together indicative of a particular signal
form a new grey-level image.
[0032] This new grey-level image is transformed into a binary image
where pixels that have grey-level values less than 100 are set to 0
and the rest of the pixels are set to.
[0033] An opening filter is applied to remove small noise particles
from the binary image and a hole filling function is applied.
[0034] The total area of the remaining cell images, which
represents the total area of infectious agent within the cell
perimeter, is recorded.
[0035] Percent area of each animal cell that is occupied by an
infectious agent can be calculated.
[0036] According to one mode of this embodiment of the invention,
detection of infected animal cells can simply be recorded for each
image or the process can be repeated for a number of optical fields
and a number of images can be accessed up to an including the
entire surface of a slide. Simple detection of infected animal
cells provides useful information for diagnosis and/or prognosis as
described in detail in Section 5.2, infra. In addition, the number
of infected animal cells can be determined to provide even more
useful information as described in Section 5.2, infra.
[0037] Alternatively, the percent area of an animal cell occupied
by an infectious agent can be calculated. This provides
quantitative information regarding extent of infection useful for
diagnosis and/or prognosis as described in detail in Section 5.2,
infra.
[0038] Additionally, according to an alternative of this mode of
the embodiment of the invention, the number and positions of all
animal cells on a slide can be determined.
[0039] According to another embodiment, an infectious agent is
detected or preferably detected and quantified by computer
controlled image analysis of a free floating infectious agent in a
sample of a body fluid or tissue from an animal. In this
embodiment, computer controlled image analysis is conducted on a
sample of body fluid or tissue treated to provide at least one
signal specific to the infectious agent of interest which can be
detected and quantified.
[0040] The computer controlled image analysis of this embodiment of
the invention is accomplished using a computer software product
including a computer-readable storage medium having fixed therein a
sequence of instructions which, when executed by a computer directs
the performance of method steps comprising:
[0041] A microscope image of an optical field of a substrate having
fixed thereon a sample of a body fluid or tissue treated to produce
a signal specific to a desired infectious agent is acquired and
transferred to the computer as an RGB image.
[0042] The image is transferred to the HLS domain.
[0043] The Hue component of the HLS image is transformed to a new
monochrome greylevel image.
[0044] The greylevel image is transformed to a "binary" image: this
is a black and white image in which pixels with pixels having
values between 10 and 25 in the Hue image are set to 255 (white)
and the rest being set to 0 black.
[0045] An "open" filter is applied: Opening is a successive
application of an "erosion" filter followed by a "dilation" filter.
It allows for the removal of small noise particles from the binary
image and a hole filling function is applied that "fills the holes"
in the identified blobs.
[0046] The area of the blobs is measured and all blobs that have an
area of less than the expected area or size of the infectious agent
are excluded from further processing. In an illustrative example,
when the infectious agent is Chlamydia, all blobs that have an area
of less than 30 pixels and more than 70 pixels are excluded from
further processing. The remaining blobs represent infectious agent
in the blood sample.
[0047] The pixel number (area) of these blobs (cells) is recorded
and saved for further processing.
[0048] The information provided by the computer controlled image
analysis methods of the present invention is useful, preferably in
combination with other information relating to the physical and/or
physiological state of a human or non-human patient in diagnosis of
prognosis of a disease or disorder associated with infection of the
human or non-human patient by an infectious agent. The information
provided is useful for diagnosis or prognosis of diseases and
disorders associated with a variety of infectious agents,
including, but not limited to, prions, bacteria, mycoplasma,
rickettsia, spirochetes, fungi, protozoal parasites, viruses,
etc.
[0049] One particular embodiment of the present invention is a
computer-implemented method for detecting a Chlamydia infected
mononuclear phagocytes from a blood or tissue sample.
[0050] Another particular embodiment of the present invention is a
computer-implemented method for detecting Helicobacter pylori
infected mononuclear phagocytes from a blood or tissue sample.
[0051] Another particular embodiment of the present invention is a
computer-implemented method for detecting CMV infected mononuclear
phagocytes from a blood or tissue sample.
[0052] Another particular embodiment of the present invention is a
computer-implemented method for detecting Herpes simplex virus
infected mononuclear phagocytes from a blood or tissue sample.
[0053] Another particular embodiment of the present invention is a
computer-implemented method for detecting mononuclear phagocytes
infected with one or more of a variety of periodontal infectious
agents such as P. gingivalis, S. sanguis, from a blood or tissue
sample.
[0054] The present invention also encompasses a computer software
product including a computer-readable storage medium having fixed
therein a sequence of instructions which, when executed by a
computer, direct the performance of steps for conducting the
methods of the invention as described herein.
[0055] The present invention also encompasses to a method of
operating a laboratory service for providing useful information for
disease diagnosis and prognosis of disease extent or susceptibility
or efficacy of a therapeutic treatment. The method encompasses the
steps of receiving a prepared substrate, e.g. a slide, that has a
monolayer of cells from a body fluid or tissue sample or receiving
a body fluid or tissue sample where the body fluid or tissue sample
is placed on a substrate as a monolayer, treating the sample to
generate a diagnostic/prognostic signal, obtaining an image of a
monolayer of cells, and operating a computerized microscope
according to the method(s) described herein to provide the
information useful to diagnose a disease or prognosticate disease
susceptibility.
[0056] The present invention also provides a system for screening
infected animal cells or infected body fluids or tissues. The basic
elements of the system include an X-Y stage, a mercury light
source, a fluorescence microscope, digital camera system such as a
color CCD camera, or a complementary metal-oxide semiconductor
(CMOS) image system, a personal computer (PC) system, and one or
two monitors and most importantly a computer software product
including a computer-readable storage medium having fixed therein a
sequence of instructions which, when executed by a computer, direct
the performance of steps for conducting the methods of the
invention as described herein. In a preferred embodiment, the stage
has an automated microscope feeder configured with it so that
slides can be automatically moved into and out of position for
image capture and/or analysis.
3.1. OBJECTS AND ADVANTAGES OF THE INVENTION
[0057] It is an object of the invention to provide methods and
systems which provide quantitative information regarding animal
(human or non-human) cells infected with an infectious agent.
Unlike other methods which provide merely for the detection of an
infectious agent, the present methods and systems advantageously
provide quantitative information with respect to infectious agents
present in animal cells. Such information, preferably, in
combination with other information relating to the physical and/or
physiological state of a human or non-human patient, is useful in
diagnosis or prognosis of a disease or disorder associated with the
presence infectious agent. Unlike other methods which merely detect
the presence of an infectious agent, in certain embodiments the
methods and systems of the present invention can provide detailed
information relating to the life cycle state and form of an
infectious agent.
[0058] It is another object of the invention to provide methods and
systems which provide quantitative information regarding the
presence of an infectious agent "free floating" in an animal human
or non-human) body fluid or tissue sample. Such quantitative
information, preferably in combination with other family/genetic
heritage, genetic profile, physical and/or physiological
information relating to the heredity, physical and/or physiological
state of a human or non-human patient, is useful in prediction of
susceptibility, diagnosis or prognosis of a disease or disorder
associated with the presence of the infectious agent.
[0059] In certain embodiments, the quantitative information
provided by the methods of the invention is used to assess
therapeutic efficacy of a treatment which the human or non-human
patient is undergoing.
[0060] In certain other embodiments, the quantitative information
provided by the methods of the invention is used to assess
susceptibility of the human or non-human animal to recurrence or
progress of a recurring or progressive disease or disorder.
[0061] In certain other embodiments, the quantitative information
provided by the methods of the invention, along with information
regarding the family history and/or genetic profile of relevant
genes of a patient is used to predict chance of onset of a disease,
advantageously prior to onset of clinical symptoms. In certain
modes of these embodiments, the information is included in a
"wellness" check or profile of the patient. For one example,
application of the methods of the invention to monitor patient
samples "over" time can be used to assess a patient's ability to
clear a bacterial infection from monocytes and hence assess
susceptibility to developing vascular disease.
BRIEF DESCRIPTION OF THE DRAWINGS
[0062] The present invention may be understood more fully by
reference to the following detailed description of the invention,
illustrative examples of specific embodiments of the invention and
the appended figures in which:
[0063] FIG. 1 is schematic representation of an apparatus or system
useful for the methods of the invention. The automated microscope
slide feeder (109) depicted in FIG. 1 is an optional component of a
preferred system useful for the methods of the invention.
[0064] FIG. 2 is a flow chart summarizing the method of one aspect
of the invention in which an infectious agent is detected or
detected and quantified in an animal cell, in FIG. 2, the
infectious agent is exemplified by Chlamydia; however, as would be
understood by those skilled in the art, any of the infectious
agents taught in this application could be detected and quantified
according to the method illustrated in FIG. 2.
[0065] FIG. 3 is a more detailed flow chart of portions of an
alternative embodiment of the method of the aspect of the invention
shown in FIG. 2.
[0066] FIG. 4 is a flow chart summarizing the method of another
aspect of the invention in which an infectious agent is detected or
detected and quantified "free floating" in a biological sample from
an animal.
DETAILED DESCRIPTION OF INVENTION
[0067] The invention will be better understood upon reading the
following detailed description of the invention and of various
exemplary embodiments of the invention, in connection with the
accompanying drawings. It will be clear to those skilled in the art
that the invention can be applied to and, in fact, encompasses
methods and systems to provide information useful in making a
disease diagnosis or a prognosis of disease or disease
susceptibility or outcome based on detection and preferably,
quantification of any characteristic resulting from infection of a
host, e.g., an animal cell with an infectious agent. The animal
cell can be obtained from any fluid or tissue sample from which it
is possible to view a monolayer of cells infected with said
infectious agent. It will also be clear to those skilled in the art
that the invention can be applied to, and in fact, encompasses
methods and systems to provide information useful in making a
disease diagnosis or prognosis of disease or disease susceptibility
or outcome based on detection, and preferably quantification, of
any characteristic indicative of the presence of an infectious
agent "free floating" in a body fluid or tissue sample.
[0068] For the purpose of this invention, information useful for
disease diagnosing or prognosis, evaluation of patient wellness or
prediction of disease onset is based upon detection of a signal
associated with a characteristic resulting from an infection of a
animal cell, tissue or body fluid. The term "characteristic" as
used herein is defined to mean any non-endogenous substance(s) that
indicates the presence of an infectious agent in a animal cell,
issue or body fluid, e.g. DNA, RNA, protein, carbohydrate,
endotoxin, etc. indicative of or specific to an infectious agent.
Fluid and tissue samples that can be used in the methods of the
invention include but are not limited to blood or a "fraction"
thereof such as white blood cells or a specific category of white
blood cells, etc., tissue biopsies, spinal fluid, meningeal fluid,
urine, alveolar fluid, etc.
[0069] In one aspect, the invention is described in connection with
observing a "monolayer" of cells. Monolayer has a specific meaning
as used herein. It means simply that the cells are arranged whereby
they are not viewed on top of one another. It does not require
confluence and can involve single cell suspensions. Thus, a
monolayer can be viewed in smears of single cell suspensions or a
layer of tissue that has a thickness of a single cell. Any solid or
exfoliative cytology technique may also be employed. Cells may be
dissociated by standard techniques known to those skilled in the
art. These techniques include but are not limited to trypsin,
collagenase or dispase treatment of the tissue.
[0070] In another aspect, the invention is described in connection
with observing a "free floating" infectious agent. Free floating
has a specific meaning as used herein. It means simply that the
infectious agent present in a body fluid or tissue is not contained
within the confines of an animal cell. It does not require that the
infectious agent "float" in a biological fluid or tissue and the
infectious agent can be attached e.g., to the periphery of a cell
or tissue. It simply is not inside an animal cell.
[0071] According to a preferred embodiment of the invention, the
animal cells are mononuclear phagocytes. As used herein, the term
"mononuclear phagocytes" is intended to comprise, without
limitation, monocytes obtained from a central or peripheral blood,
macrophages obtained from any site, including any tissue or cavity,
macrophages derived by incubating or culturing macrophage
precursors obtained from bone marrow or blood. According to a more
preferred embodiment, the mononuclear phagocytes are obtained from
peripheral blood samples.
[0072] For the purpose of this invention, an "infectious agent" is
intended to mean an organism or other agent, capable of replication
and associated with a disease or disorder when said agent infects a
host, such as an animal host or a plant host. Infectious agents
include, but are not limited to, prions, bacteria, mycoplasma,
rickettsia, spirochetes, fungi, parasites, protozoa, viruses,
etc.
[0073] In an important embodiment, the infectious agent is a
Chlamydia species. Common Chlamydia include: C. pneumoniae, C.
trachomatis, C. psittaci and C. pecorum.
[0074] In another important embodiment, the infectious agent is a
Helicobacter species. Common Helicobacter include: H. pylori and H.
felis.
[0075] In another important embodiment, the infectious agent is a
Borrelia species. Common Borrelia include: B. burgdorferi and B.
recurrentis.
[0076] In another important embodiment, the infectious agent is a
common periodontal bacteria. Common periodontal bacteria include:
P. gingivalis, and P. sanguis.
[0077] In yet another important embodiment, the infectious agent is
a Herpes virus, such as Herpes simplex virus or
Cytomegalovirus.
[0078] Solely for ease of explanation, the description of the
invention is divided into the following sections: (1) methods and
systems (including apparatus) for detection and quantification of
an infectious agent, including quantification of an infectious
agent (a) in an animal cell and (b) "free floating" in a body fluid
or tissue sample: and (2) applications and diseases and disorders
for which useful information is provided.
5.1. METHODS AND SYSTEMS FOR DETECTION OR DETECTION AND
QUANTIFICATION
[0079] The present invention provides methods, apparatus and
systems for the rapid detection and, more preferably, detection and
quantification of an infectious agent in a biological sample. The
methods employ apparatus and systems which afford computer
controlled automated image analysis.
[0080] FIG. 1 illustrates the basic elements of a system suitable
for use according to the methods of the invention. A system such as
illustrated in FIG. 1 can be used in any of die methods described
in the sub-sections below. The basic elements of the system include
an X-Y stage (101), a mercury light source (102), a fluorescence
microscope (103), a color CCD camera or CMOS image sensor (105), a
personal computer (PC) system (106), and one or two monitors (107
and 108). In a preferred embodiment, the stage has an automated
microscope feeder (109) configured with it so that slides can be
automatically moved into and out of position for image capture
and/or analysis.
[0081] The individual elements of the system can be custom built or
purchased off-the-shelf as standard components. Each element is
described in somewhat greater detail below.
[0082] The X-Y stage can be any motorized positional stage suitable
for use with the selected microscope. Preferably, the X-Y stage can
be a motorized stage that can be connected to a personal computer
and electronically controlled using specifically compiled software
commands. When using such an electronically controlled X-Y stage, a
stage controller circuit card plugged into an expansion bus of the
PC connects the stage to the PC. The stage should also be capable
of being driven manually. Electronically controlled stages such as
described here are produced by microscope manufacturers, for
example including Olympus (Tokyo, Japan), as well as other
manufacturers, such as LUDL (NY, USA).
[0083] The microscope can be any fluorescence microscope equipped
with a reflected light fluorescence illuminator and a motorized
objective lens turret with a 20.times., 40.times., and 100.times.
(with or without oil immersion) objective lens, providing a maximum
magnification of 1000.times. The motorized nosepiece is preferably
connected to the PC and electronically switched between successive
magnifications using specifically compiled software commands. When
using such an electronically controlled motorized nosepiece, a
nosepiece controller circuit card plugged into an expansion bus of
the PC connects the stage to the PC. The microscope and stage are
set up to include a mercury light source, capable of providing
consistent and substantially even illumination of the complete
optical field.
[0084] The microscope produces an image viewed by the camera. The
camera can be any color 3-chip CCD camera or other camera or
digital image sensor system connected to provide an electronic
output and providing high sensitivity and resolution. The output of
the camera is fed to a frame grabber and image processor circuit
board installed in the PC. A camera found to be suitable is the
OPTRONICS 750 (OPTRONICS, CA.). Another digital camera found to be
suitable is the complementary metal-oxide semiconductor system
(CMOS) image sensor available from Photobit (Pasadena, Calif.).
[0085] Various frame grabber systems can be used in connection with
the present invention. The frame grabber can be, for example, the
MATROX GENESIS available from MATROX (Montreal, CANADA). The MATROX
GENESIS module features on-board hardware supported image
processing capabilities. These capabilities compliment the
capabilities of the MATROX IMAGING LIBRARY (MIL) software package.
Thus, it provides extremely fast execution of the MIL based
software algorithms. The MATROX boards support display to a
dedicated SVGA monitor. The dedicated monitor is provided in
addition to the monitor usually used with the PC system. Any
monitor SVGA monitor suitable for use with the MATROX image
processing boards can be used. One dedicated monitor usable in
connection with the invention is a ViewSonic 4E (Walnut Creek,
Calif.) SVGA monitor.
[0086] In order to have sufficient processing and storage
capabilities available, the PC can be any PC such as an INTEL
PENTIUM-based PC having at least 256 MB RAM and at least 40 GB of
hard disk drive storage space. The PC preferably further includes a
monitor. Other than the specific features described herein, the PC
is conventional, and can include keyboard, printer or other desired
peripheral devices not shown.
[0087] Alternatively, automated sample analysis may be performed by
an apparatus and system for distinguishing, in an optical field,
objects of interest from other objects and background, such as the
automated system exemplified in U.S. Pat. No. 5,352,613, issued
Oct. 4, 1994 (incorporated herein by reference). Furthermore, once
an object, i.e., a relevant animal cell has been identified, the
color, e.g. the combination of the red, green, blue components for
the pixels that comprise the object, or other parameters of
interest relative to that object, i.e., whether it is infected or
not by an infectious agent, can be measured and stored.
[0088] Other examples of alternative apparatus and systems for
automated sample analysis are illustrated in WO99/58972 and
PCT/US00/31494 (incorporated herein by reference).
[0089] One suitable system consists of an automatic microscopical
sample inspection system having:
[0090] a sample storage module and loading and unloading module
[0091] a sample transporting mechanism to and from an automated
stage that moves the sample under a microscope objective lenses
array
[0092] an array of CCD cameras
[0093] a processing unit having a host computer, multiple
controllers to control all mechanical parts of the microscopy
system and
[0094] a high speed image processing unit where the CCD cameras are
connected (see, PCT/US00/31494).
[0095] An innovative feature of this embodiment of a computer
controlled system is an array of two or more objective lenses
having the same optical characteristics. The lenses are arranged in
a row and each of them has its own z-axis movement mechanism, so
that they can be individually focused. This system can be equipped
with a suitable mechanism so that the multiple objective holder can
be exchanged to suit the same variety of magnification needs that a
common single-lens microscope can cover. Usually the magnification
range of light microscope objectives extends from 1.times. to
100.times.
[0096] Each objective is connected to its own CCD camera. The
camera field of view characteristics are such that it acquires the
full area of the optical field as provided by the lens.
[0097] Each camera is connected to an image acquisition device.
This is installed in a host computer. For each optical field
acquired, the computer is recording its physical location on the
microscopical sample. This is achieved through the use of a
computer controlled x-y mechanical stage. The image provided by the
camera is digitized and stored in the host computer memory. With
the current system, each objective lens can simultaneously provide
an image to the computer, each of which comprises a certain portion
of the sample area. The lenses should be appropriately corrected
for chromatic aberrations so that the image has stable qualitative
characteristics all along its area.
[0098] The imaged areas will be in varying physical distance from
each other. This distance is a function of the distance at which
the lenses are arranged and depends on the physical dimensions of
the lenses. It will also depend on the lenses' characteristics,
namely numerical aperture and magnification specifications, which
affect the area of the optical field that can be acquired.
Therefore, for lenses of varying magnification/numerical aperture,
the physical location of the acquired image will also vary.
[0099] The computer will keep track of the features of the
objectives-array in use as well as the position of the motorized
stage. The stored characteristics of each image can be used in
fitting the image in its correct position in a virtual patchwork,
e.g. "composed" image, in the computer memory.
[0100] The host computer system that is controlling the above
configuration, is driven by software system that controls all
mechanical components of the system through suitable device
drivers. The software also comprises properly designed image
composition algorithms that compose the digitized image in the
computer memory and supply the composed image for processing to
further algorithms. Through image decomposition, synthesis and
image processing specific features particular to the specific
sample are detected.
5.1.1. METHODS FOR DETECTION OR DETECTION AND QUANTIFICATION OF
AGENT INSIDE AN ANIMAL CELL
[0101] According to one embodiment, an infectious agent is detected
or preferably detected and quantified by computer controlled image
analysis of animal cells in a sample of a body fluid or tissue from
an animal. Using this method, animal cells infected with an
infectious agent are detected or preferably detected and
quantified.
[0102] According to this embodiment of the method of the invention,
computer controlled image analysis is conducted on a sample of body
fluid or tissue containing animal cells in a monolayer treated to
provide at least two different signals which can both be detected
and quantified. At least two signals are required. A first signal
is employed to identify an animal cell of interest and a second
signal is employed to identify an infectious agent within an
identified animal cell of interest. Detection and quantification of
the two different signals provides for determination of: (1) the
number of infected animal cells, e.g., per unit volume of body
fluid, (2) the number of infectious agents per animal cell or (3)
both the number of infected animal cells and the number of
infectious agents per cell, i.e., the extent of cell infection.
[0103] As used herein, "signal" should be taken in its broadest
sense, as a physical manifestation which can be detected and
identified, thus carrying information. One simple and useful signal
is the light emitted by a fluorescent dye selectively bound to a
structure of interest. That signal indicates the presence of the
structure, i.e., either an animal cell or an infectious agent,
which might be difficult to detect absent the fluorescent dye.
[0104] The requirements and constraints on the generation of the
first signal and second signal are relatively simple. The materials
and techniques used to generate the first signal should not
interfere adversely with the materials and techniques used to
generate the second signal (to an extent which compromises
unacceptably detection and quantification), and visa versa. Nor
should they damage or alter the cell- or infectious agent-specific
characteristics sought to be measured to an extent that compromises
unacceptably the detection and quantification. Finally, any other
desirable or required treatment of the cells should also not
interfere with the materials or techniques used to generate the
first signal and second signal to an extent that compromises
unacceptably the detection and quantification. Within those limits,
any suitable generators of the first signal and second signal may
be used.
[0105] According to the method, a monolayer of animal cells, fixed
to a suitable solid substrate is observed by a computer controlled
microscope system as described above. The monolayer can be obtained
merely by spreading a body fluid or tissue sample with animal cells
of interest on a solid substrate, such as a slide. Alternatively, a
monolayer can be obtained by spreading a sample containing an
enriched population of animal cells of interest on a solid
substrate. Enrichment of animal cells of interest can be
accomplished by any means known to those skilled in the art, such
as centrifugation, affinity purification, etc. A physical feature
of the animal cells can be used to provide a first signal, or more
preferably the animal cells are stained to produce a first signal.
The fixed animal cells are also treated to produce a second signal
specific to an infectious agent, if said agent is present.
Optionally, the method is conducted using animal cells already
fixed on a substrate, e.g. a slide. Alternatively, as part of the
method of the present invention, the cells may be prepared on a
substrate before being treated. Preparing the cells may be
accomplished by first obtaining a sample, washing the cells, and
fixing the cells on a substrate. It should be evident that any
means of obtaining, collecting, washing, and fixing the cells may
be used depending on the infected animal cell to be detected.
[0106] The body fluid or tissue sample may be obtained by a number
of ways depending upon where the infected cell resides. If, for
example, the infected cell resides in blood, a minimally invasive
procedure, such as taking a blood sample, may be all that is
necessary to obtain the cells. As an illustrative example,
Chlamydia infected mononuclear phagocytes can be observed and
quantitated in a peripheral blood sample. A similar minimally
invasive procedure may be used if the infected cell resides in a
mucosa layer. In an alternative embodiment of the present
invention, a sample may be taken by a more invasive procedure if
desired. For example, a biopsy may be performed in order to obtain
a sample to identify an infected animal cell needed to make a
diagnosis or prognosis with the present invention.
[0107] Treating the cells may occur with a fluorescent dye bound to
an antibody against the characteristic that specifically identifies
the infectious agent. The animal cells may be simply counterstained
with an agent that fluoresces at a wavelength different from that
of the fluorescent dye bound to the antibody specific for the
infectious agent. Alternatively, animal cells are treated with a
fluorescent dye bound to an antibody specific for the animal cells
which dye fluoresces at a wavelength different from that of the dye
bound to the antibody specific for the infectious agent. It should
now be evident that any two detectable indicators of the presence
of an infected animal cell may serve as the first and second
signals, subject to certain constraints noted herein.
[0108] As would be understood by those skilled in the art, for each
infectious agent to be detected, specific reagents are required to
generate the signals needed to detect the infected animal cell. For
example, for the embodiment in which Chlamydia infected mononuclear
phagocytes are detected, a labelled antibody directed to Chlamydia
can be used in generating the signal. Three examples of
commercially available antibodies are: (1) ChlamydiaCel, a
genus-specific lipopolysaccharide monoclonal antibody that
identifies C. pneumoniae, as well as C. trachomatis and C.
psittaci, with a strong fluorescence of the intracellular
inclusions, the pinhead-sized elementary bodies and the free
cell-associated Chlamydia LPS antigens (CelLabs, Sydney,
Australia), (2) Chlamydia CelPn, a C. pneumoniae specific
monoclonal antibody (CelLabs, Sydney, Australia), and (3) a
Chlamydia specific FITC-conjugated monoclonal antibody (Kallestad
Diagnostics, Chaska, Minn.). Other illustrative examples of
commercially available antibodies specific to cells of interest or
infectious agents of interest that are useful in the methods and
systems of the invention include: Clone MCA38, mouse monoclonal
antibody to human monocytes/macrophages (Research Diagnostics Inc.,
Flanders, N.J.); Clone B2, monoclonal antibody for the detection of
cytomegalovirus (Research Diagnostics Inc., Flanders, N.J.); Clone
A33, monoclonal antibody for the detection of herpes simplex virus
(Research Diagnostics Inc., Flanders, N.J.); Antibody 05-97-92,
affinity purified antibody to Borrelia species (Kirkegaard and
Perry Laboratories, Gaithersburg, Md.); and Monoclonal antibody
A94000136P, to Helicobacter pylori, (BiosPacific, Emeryville,
Calif.).
[0109] As would be understood by those skilled in the art,
antibodies or antibody fragments specific for any host cell of
interest or any infectious agent can be produced by any method
known in the art for the synthesis of antibodies (or binding
fragments thereof, in particular, by chemical synthesis or
preferably, by recombinant expression techniques.
[0110] Polyclonal antibodies to a host cell of interest or in
infectious agent of interest can be produced by various procedures
well known in the art. For example, a host cell of interest, an
infectious agent or antigen thereof can be administered to various
host animals including, but not limited to, rabbits, mice, rats,
etc. to induce the production of sera containing polyclonal
antibodies specific for the infectious agent or host cell or
antigen thereof. Various adjuvants may be used to increase the
immunological response, depending on the host species, and include
but are not limited to, Freund's (complete and incomplete), mineral
gels such as aluminum hydroxide, surface active substances such as
lysolecithin, pluronic polyols, polyanions, peptides, oil
emulsions, keyhole limpet hemocyanins, dinitrophenol, and
potentially useful human adjuvants such as BCG (bacille
Calmette-Guerin) and corynebacterium parvum. Such adjuvants are
also well known in the art.
[0111] Monoclonal antibodies can be prepared using a wide variety
of techniques known in the art including the use of hybridoma,
recombinant, and phage display technologies, or a combination
thereof. For example, monoclonal antibodies can be produced using
hybridoma techniques including those known in the art and taught,
for example, in Harlow et al., Antibodies: A Laboratory Manual,
(Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et
al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681
(Elsevier, N.Y., 1981) (said references incorporated by reference
in their entireties). The term "monoclonal antibody" as used herein
is not limited to antibodies produced through hybridoma technology.
The term "monoclonal antibody" refers to an antibody that is
derived from a single clone, including any eukaryotic, prokaryotic,
or phage clone, and not the method by which it is produced.
[0112] Methods for producing and screening for specific antibodies
using hybridoma technology are routine and well known in the art.
Briefly, mice can be immunized with a host cell of interest or
antigenic part thereof, infectious agent or antigen thereof and
once an immune response is detected, e.g., antibodies specific for
the host cell of interest or infectious agent are detected in the
mouse serum, the mouse spleen is harvested and splenocytes
isolated. The splenocytes are then fused by well known techniques
to any suitable myeloma cells, for example cells from cell line
SP20 available from the ATCC. Hybridomas are selected and cloned by
limited dilution. The hybridoma clones are then assayed by methods
known in the art for cells that secrete antibodies capable of
binding an infectious agent or host cell of interest. Ascites
fluid, which generally contains high levels of antibodies, can be
generated by immunizing mice with positive hybridoma clones.
[0113] Antibody fragments which recognize specific epitopes of a
host cell or of an infectious agent may be generated by any
technique known to those of skill in the art. For example, Fab and
F(ab')2 fragments may be produced by proteolytic cleavage of
immunoglobulin molecules, using enzymes such as papain (to produce
Fab fragments) or pepsin (to produce F(ab')2 fragments). F(ab')2
fragments contain the variable region, the light chain constant
region and the CHI domain of the heavy chain. Further, the
antibodies useful to generate a signal (e.g., when labelled) can
also be generated using various phage display methods known in the
art.
[0114] In phage display methods, functional antibody domains are
displayed on the surface of phage particles which carry the
polynucleotide sequences encoding them. In particular, DNA
sequences encoding VH and VL domains are amplified from animal cDNA
libraries (e.g., human or murine cDNA libraries of lymphoid
tissues). The DNA encoding the VH and VL domains are recombined
together with an scFv linker by PCR and cloned into a phagemid
vector (e.g., p CANTAB 6 or pComb 3 HSS). The vector is
electroporated in E. coli and the E. coli is infected with helper
phage. Phage used in these methods are typically filamentous phage
including fd and M13 and the VH and VL domains are usually
recombinantly fused to either the phage gene III or gene VIII.
Phage expressing an antigen binding domain that binds to a host
cell or an infectious agent of interest can be selected or
identified with a host cell infectious agent or antigen thereof,
e.g., using labeled antigen or antigen bound or captured to a solid
surface or bead. Examples of phage display methods that can be used
to make the antibodies useful in the method of the present
invention include those disclosed in Brinkman et al., 1995, J.
Immunol. Methods 182:41-50; Ames et al., 1995, J. Immunol. Methods
184:177-186; Kettleborough et al., 1994, Eur. J. Immunol.
24:952-958; Persic et al., 1997, Gene 187:9-18; Burton et al.,
1994, Advances in Immunology 57:191-280: PCT application No.
PCT/GB91/O1 134; PCT publication Nos. WO 90/02809, WO 91/10737, WO
92/01047, WO 92/18619, WO 93/1 1236, WO 95/15982, WO 95/20401, and
WO97/13844; and U.S. Pat. Nos. 5,698,426, 5,223,409, 5,403,484,
5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908,
5,516,637, 5,780,225, 5,658,727, 5,733,743 and 5,969,108; each of
which is incorporated herein by reference in its entirety.
[0115] As described in the above references, after phage selection,
the antibody coding regions from the phage can be isolated and used
to generate whole antibodies or any other desired antigen binding
fragment, and expressed in any desired host, including mammalian
cells, insect cells, plant cells, yeast, and bacteria, e.g., as
described below. Techniques to recombinantly produce Fab, Fab' and
F(ab').sub.2 fragments can also be employed using methods known in
the art such as those disclosed in PCT publication No. WO 92/22324;
Mullinax et al., 1992, BioTechniques 12(6):864-869; Sawai et al.,
1995, AJRI 34:26-34; and Better et al., 1988, Science 240:1041-1043
(said references incorporated by reference in their
entireties).
[0116] To generate whole antibodies, PCR primers including VH or VL
nucleotide sequences, a restriction site, and a flanking sequence
to protect the restriction site can be used to amplify the VH or VL
sequences in scFv clones. Utilizing cloning techniques known to
those of skill in the art, the PCR amplified VH domains can be
cloned into vectors expressing a VH constant region, e.g., the
human gamma 4 constant region, and the PCR amplified VL domains can
be cloned into vectors expressing a VL constant region, e.g., human
kappa or lamba constant regions. Preferably, the vectors for
expressing the VII or VL domains comprise an EF-1.alpha. promoter,
a secretion signal, a cloning site for the variable domain,
constant domains, and a selection marker such as neomycin. The VH
and VL domains may also cloned into one vector expressing the
necessary constant regions. The heavy chain conversion vectors and
light chain conversion vectors are then co-transfected into cell
lines to generate stable or transient cell lines that express
full-length antibodies, e.g., IgG, using techniques known to those
of skill in the art.
[0117] Alternatively, the second signal, which indicates the
presence of a particular characteristic of an infectious agent
being tested for, and may be generated, for example, using
technologies in which nucleic acids specific to the infectious
agent are labelled and detected using techniques including such as
in situ PCR-amplification or PCR in situ hybridization or FISH.
[0118] Cells that emit both signals, e.g. the cell is an infected
animal cell and contains the characteristic being tested for with
the second signal, are scored. Counts may be maintained of the
number and strengths of the first signal and second signal
detected.
[0119] While the description herein explains the method with
respect to mononuclear phagocytes (or monocytes) as the host, e.g.,
animal cells; blood as the body fluid or tissue sample and
Chlamydia as the infectious agent, it would be understood by those
skilled in the art that the invention can be applied to and in
fact, encompasses computer controlled automated image analysis of
any type of host cell and any body fluid or tissue sample infected
by any relevant infectious agent. Hence, it is understood that the
following description is merely explained, for purposes of ease of
explanation and is illustrated in FIGS. 2 and 3, with respect to
detection and quantification of mononuclear phagocytes infected
with Chlamydia.
[0120] FIG. 2 schematically illustrates one embodiment of one
method of the invention, using monocytes as the animal cells and
Chlamydia as the infectious agent, merely for ease of
explanation.
[0121] An objective, e.g., the 100.times. objective is selected to
provide total magnification of 1000.times. and a slide containing a
monolayer of treated animal cells (from an animal suspected of
infection with Chlamydia) is placed on the automated stage.
[0122] As illustrated in FIG. 2, a slide is loaded (200),
(preferably using an automated slide loader.
[0123] The program moves the automated stage to a preset starting
point, e.g., on a corner of the slide.
[0124] Optionally, the x-y position of the stage at the starting
point is recorded.
[0125] An optical field is selected and the microscopic image is
acquired and transferred to the computer as an RUB image (201).
[0126] The Red component of the RGB image is transferred to a new
monochrome grey-level image and clipped for pixel values of less
than 50 to cut down signal noise (202).
[0127] The grey-level image is transformed to a binary image, a
black and white image in which pixels with corresponding pixels in
the Luminance image having grey-level values lower than the cut off
point are set to 255 (white) (203).
[0128] Noise particles are removed and a hole filling function is
applied to the binary image (204). In an illustrative embodiment,
(see infra Section 6) a commercially available MATROX function is
used to fill the holes in the identified images. The area of each
image is measured and all images having an area of expected for a
monocyte or mononuclear phagocyte, i.e., 1000 pixels or greater are
selected as representative of mononuclear phagocytes. Cell images
that have an area less than 1000 pixels are excluded from further
processing (205).
[0129] The area, in pixels, of the cell images, i.e., mononuclear
phagocytes, is recorded and saved for further processing (206).
Optionally, the XY location of mononuclear phagocytes is recorded
for later analysis and/or processing.
[0130] The original Red component from the original RGB image is
transferred to a binary image, so that pixels having grey-level
values less than 200 are set to 0 while all the rest are set to 255
(207a).
[0131] The original Green component is transferred to grey level
image (207b).
[0132] All pixels that have a value of M and any grey-level value
in the Green component of the original RUB image equal to N form a
new grey-level image (208). In a specific illustrative embodiment
exemplified infra in Section 6 when a first signal indicative of a
monocyte is generated using counter stain with
4'-6-Diamidino-2-phenylindole is detected and a second signal
indicative of Chlamydia is detected using fluorophore conjugated
ChlamydiaCel monoclonal antibody available from (CelLabs, Sydney,
Australia) is used, M is set at 255 and N is set at 0.
[0133] This new grey-level image is transformed into a binary image
where pixels that have grey-level values less than 100 are set 0
and the rest of the pixels are set to 255 (209).
[0134] If the level is 255, the monocyte is counted and recorded as
an Chlamydia-infected monocyte (212); if not, the monocyte is
recorded as not infected (211).
[0135] The extent of infection of individual Chlamydia infected
monocytes can be determined:
[0136] An opening filter is applied to remove small noise particles
from the binary image and a hole filling function is applied
(214)
[0137] The total area of the remaining cell images, which
represents the total area of Chlamydia organisms within the cell
perimeter is recorded (215)
[0138] Percent area of each mononuclear phagocyte that is occupied
by Chlamydia organisms can be calculated (216).
[0139] As illustrated in FIG. 2, according to one mode of this
embodiment of the invention, detection of Chlamydia infected
monocytes can simply be recorded for each image (210-212) or the
process can be repeated for a number of optical fields and a number
of images can be accessed (218) up to an including the entire
surface of a slide (218-217). Simple detection of infected
monocytes provides useful information for diagnosis and/or
prognosis as described in detail in Section 5.2, infra. In
addition, the number of infected monocytes can be determined to
provide even more useful information as described in Section 5.2,
infra.
[0140] Alternatively, as further illustrated in FIG. 2, the percent
area of a monocyte occupied by Chlamydia is calculated (213-216).
This provides quantitative information regarding extent of
infection useful for diagnosis and/or prognosis as described in
detail in Section 5.2, infra.
[0141] FIG. 3 schematically illustrates an alternative mode of this
embodiment of the invention. As illustrated in FIG. 3, according to
an alternative of this mode of the embodiment of the invention, the
number and positions of all monocytes on a slide are determined. In
this mode of this embodiment of the present invention, Chlamydia
infection of monocytes can be detected or detected and quantified
using the steps illustrated in FIG. 2 either after recordation of
each monocyte image (see FIG. 3, 307) or after all monocyte images
have been recorded (see FIG. 3, 309). This provides information
useful as described in Section 5.2, infra.
[0142] As would be understood by those skilled in the art, the
parameters to set levels of image detection for animal cells and
for infectious agents will depend upon the specific animal cells
and infectious agents to be detected as well as upon the specific
first and second signals used to detect the cells and infectious
agents. Such parameters can be determined by those skilled in the
art using known methods and the information provided herein.
5.1.2. METHODS FOR DETECTION OR DETECTION AND QUANTIFICATION OF A
FREE-FLOATING AGENT
[0143] According to another embodiment, an infectious agent is
detected or preferably detected and quantified by computer
controlled image analysis of a free floating infectious agent in a
sample of a body fluid or tissue from an animal. In this
embodiment, computer controlled image analysis is conducted on a
sample of body fluid or tissue treated to provide at least one
signal specific to the infectious agent of interest which can be
detected and quantified. As would be understood by those skilled in
the art, the same signals specific for an infectious agent
described in Section 5.1.1., supra, are suitable for use in this
embodiment of the invention.
[0144] A slide containing, for example, a blood smear from a
patient suspected of infection with Chlamydia is properly
immunostained, for example, using the following steps: blood smears
are washed with phosphate-buffered saline, fixed with methanol and
stained directly with Chlamydia genus-specific FITC-conjugated
monoclonal antibody obtained e.g. from (Kallestad Diagnostics,
Chaska, Minn.).
[0145] Images are captured at 1000.times. total magnification. The
PC (see FIG. 1, 106) software program compiled in MICROSOFT C++
using the MATROX IMAGING LIBRARY (MIL). MIL is designed by MATROX
to control the operation of the frame grabber and the processing of
images captured by frame grabber and subsequently stored in PC as
disk files. It comprises a number of image processing specialized
routines particularly suitable for performing such image processing
task as filtering, object selection and various measurement
functions. The program prompts and measurement results are shown on
the computer monitor (see FIG. 1, 108).
[0146] FIG. 4 schematically illustrates this embodiment of the
method of the invention, using Chlamydia as the infectious agent,
merely for illustrative purposes and ease of explanation.
[0147] As illustrated in FIG. 4, detection or detection and
quantification of free floating Chlamydia in a blood sample on a
microscope slide is achieved as follows:
[0148] An optical field is selected and the microscopic image is
acquired and transferred to the computer as an ROB image (401);
[0149] The image is transferred in the HLS domain (402);
[0150] The Hue component of the HLS image is transformed to a new
monochrome grey-level image (403);
[0151] A "binary" image is formed: this is a black and white image
in which pixels with pixels having values between 10 and 25 in the
Hue image set to 255 (white) and the rest being set to 0 black
(404);
[0152] An "open" filter is applied: Opening is a successive
application of an "erosion" filter followed by a "dilation" filter.
It allows for the removal of small noise particles from the binary
image. A hole filling function is applied. (405);
[0153] The area of the blobs is measured and all blobs that have an
area of less than 30 pixels and more than 70 are excluded from
further processing. The remaining blobs represent identified
Chlamydia particles in the blood sample (406);
[0154] The pixel number (area) of these blobs (cells) is recorded
and saved for further processing (407).
5.2. DISEASES, DISORDERS AND APPLICATIONS
[0155] The information provided by the computer controlled image
analysis methods of the present invention is useful, preferably in
combination with other information relating to the
genetic/hereditary, physical and/or physiological state of a human
or non-human patient, in diagnosis or prognosis of a disease or
disorder associated with infection of the human or non-human
patient by an infectious agent. Prognosis encompasses determination
of the susceptibility to disease onset or progression, spread or
recurrence, of extent of disease or of whether a treatment a
patient is undergoing is effective in ameliorating, preventing or
curing a disease or disorder from which the patient is suffering.
The information provided is useful for diagnosis or prognosis of
diseases and disorders associated with a variety of infectious
agents, including, but not limited to, prions, bacteria,
mycoplasma, rickettsia, spirochetes, fungi, protozoal parasites,
viruses, etc.
[0156] In certain embodiments, the methods provide information
useful and sufficient to diagnose a disease or disorder caused by
an infectious agent. As an illustrative example, the methods of the
invention are used to detect or detect and quantitate the presence
of Chlamydia pneumoniae in a body fluid or tissue sample to
diagnose the presence of pneumonia caused by said infectious agent.
As another illustrative example the methods of the invention are
used to detect and quantitate the presence of Borrelia in a body
fluid or tissue sample to diagnose the presence of Lyme disease
caused by said infectious agent.
[0157] Thus, in certain embodiments, simple detection of the
presence of an infectious agent either in an animal cell or free
floating in a body fluid or tissue provides sufficient information
to diagnose a disease caused by the infectious agent. In other
embodiments, quantitative information regarding the extent or
presence of an infectious agent, e.g. a virus is useful for
prognosis of a disease cause by the infectious agent.
[0158] in yet other embodiments, the quantitative information
regarding an infectious agent provided by the methods of the
invention is used to determine therapeutic efficacy of a treatment
which a patient infected with an infectious agent is
undergoing.
[0159] In an important specific embodiment, the invention provides
is a unique method to detect mononuclear phagocytes infected with
Chlamydia, Helicobacter or a limes virus to provide information
useful for diagnosis or prognosis of vascular disease, such as
atherosclerosis or arteriolosclerosis or coronary disease, such as
congestive heart failure or susceptibility to myocardial
infarction. Rather than identifying vascular disease, such as
atherosclerosis, with invasive procedures by viewing the
manifestations of fatty streaks, intermediate lesions, or fibrous
plaques, this embodiment of the present invention provides
information useful for determination of atherosclerosis or
susceptibility to same using a minimally-invasive procedure by
analysis of a blood sample, e.g., a peripheral blood sample.
Determination of a change, especially an increase in the number of
infected monocytes is important for a prognosis of disease since
published epidemiological studies of vascular and coronary heart
disease show at least a two-fold or larger odds ratio with the
presence of infected monocytes. Some show increasing odds ratios
with increasing antibody titres.
[0160] In another specific embodiment, the invention provides a
unique method to detect and preferably quantitate the presence of
an infectious agent such as Chlamydia in blood, spinal fluid or
other body fluid. This information is particularly useful in
determining the etiology of certain diseases or disorders of the
central nervous system.
6. EXEMPLARY EMBODIMENT
[0161] The following non-limiting example illustrates the detection
of Chlamydia pneumoniae infected mononuclear phagocytes to provide
information useful for prognosticating atherosclerosis
susceptibility. In this example, a peripheral blood sample is
obtained from a patient suffering from at least one of the signs
and symptoms of atherosclerosis. Using the automated image analysis
method of the present invention, quantitative determination is made
of the extent of Chlamydia infection of the patient's monocytes.
Based on the information provided, a determination can be made
regarding whether or not a course of treatment with antibiotic(s)
effective against Chlamydia would be useful for ameliorating or
preventing progression of at least one symptom or sign of
atherosclerosis.
6.1. SMEAR PREPARATION
[0162] A non-coagulated whole blood sample is subjected to
centrifugation through a Ficoll-Histopaque density gradient. The
fraction containing mononuclear phagocytes is collected and washed
with phosphate-buffered saline. Smears are prepared from aliquots
of cells on glass microscope slides.
6.1.1. CELL FIXATION
[0163] Smears are fixed in methanol for 5-10 minutes.
Alternatively, smears may be fixed in ice-cold 0.05% glutaraldehyde
for 10-30 minutes at room temperature. The smears are then washed
with phosphate-buffered saline containing 0.1% bovine serum
6.1.2. CELL STAINING
[0164] Staining for C. pneumoniae infected mononuclear phagocytes
entails incubating the cells with fluorophore-conjugated
genus-specific anti-Chlamydial monoclonal antibody for 15-60
minutes at room temperature in the dark. For example, ChlamydiaCel
(available from CelLabs, Sydney, Australia) can be used. The smear
then is counterstained with 4',6-Diamidino-2-phenylindole ("DAPI")
for 5 minutes at room temperature. The smear is washed with
phosphate-buffered saline and allowed to air dry.
6.2. AUTOMATED SMEAR ANALYSIS
[0165] C. pneumoniae infected mononuclear phagocytes are identified
to provide inform useful to prognosticate atherosclerosis
susceptibility. Automated image analysis of the smear is conducted
as described in Section 5.1.1, supra. A description of the detailed
method used in the exemplary embodiment follows.
6.2.1. METHOD
[0166] The PC executes a smear analysis software program compiled
in MICROSOFT C++ using the MATROX IMAGING LIBRARY (MIL). MIL is a
software library of functions, including those which control the
operation of the frame grabber and which process images captured by
the frame grabber for subsequent storage in PC as disk files. MIL
comprises a number of specialized image processing routines
particularly suitable for performing such image processing tasks as
filtering, object selection and various measurement functions. The
smear analysis software program runs as a WINDOWS 95 application.
The program prompts and measurement results are shown on the
computer monitor, while the images acquired through the imaging
hardware are displayed on the dedicated imaging monitor.
6.2.2. DETECTION OF THE SIGNAL
[0167] The Chlamydia infected mononuclear phagocyte detection
algorithm is illustrated in the flow charts of FIGS. 2 and 3, where
possible cell positions are identified at 1000.times. total
magnification. The 100.times. objective is selected and the search
for C. pneumoniae infected mononuclear phagocyte is conducted:
[0168] An optical field is selected and the microscopic image is
acquired and transferred to the computer as an RGB image (201).
[0169] The Red component of the RGB image is transferred to a new
monochrome grey-level image and clipped for pixel values of less
than 50 to cut down signal noise (202).
[0170] The grey-level image is transformed to a binary image, a
black and white image in which pixels with corresponding pixels in
the Luminance image having grey-level values lower than the cut off
point are set to 255 (white) (203).
[0171] An opening filter, successive applications of an erosion
filter followed by a dilation filter, is applied for the removal of
small noise particles from the binary image (204).
[0172] Application of a hole filling function fills the holes in
the identified images (204). In this illustrative example, the hole
filling function is commercially available as a MATROX function
from Matrox (Montreal, Canada).
[0173] The area of each image is measured and all images having an
area of 1000 pixels or greater are representative of mononuclear
phagocytes. Cell images that have an area less than 1000 pixels are
excluded from further processing (205).
[0174] The area, in pixels, of the cell images, i.e., mononuclear
phagocytes, is recorded and saved for further processing (206).
[0175] The original Red component from the original RGB image is
transferred to a binary image, so that pixels having grey-level
values less than 200 are set to 0 while all the rest are set to 255
(207a).
[0176] The original Green component is transferred to grey level
image (207b).
[0177] All pixels that have a value of 255 and any grey-level value
in the Green component of the original RGB image form a new
grey-level image (208).
[0178] This new grey-level image is transformed into a binary image
where pixels that have grey-level values less than 100 are set 0
and the rest of the pixels are set to 255 (209).
[0179] The monocyte can be counted and recorded as an uninfected or
Chlamydia-infected monocyte (210-212), or
[0180] An opening filter is applied to remove small noise particles
from the binary image (214).
[0181] The total area of the remaining cell images, which
represents the total area of Chlamydia organisms with in the cell
perimeter, is recorded. (215).
[0182] Percent area of each mononuclear phagocyte that is occupied
by Chlamydia organisms can be calculated (216).
[0183] As further illustrated in FIG. 2, all optical fields on a
slide can be assessed (FIG. 2, 217-118). The algorithm is repeated
for additional fields of the smear according to the flow chart of
FIG. 3. After Chlamydia infected mononuclear phagocytes are
identified and quantified, the quantitative information is used, in
combination with other relevant information regarding the physical
and physiological state of the patient's vascular system to
determine whether a course of antibiotic treatment is indicated for
the patient.
[0184] Computer and image processing technologies are constantly
changing. Newer technologies which meet the needs of the
above-described methods and apparatus, while not specifically
described here, are clearly contemplated as within the invention.
For example, certain conventional pixel and image file formats are
mentioned above, but others may also be used. Image files may be
compressed using JPEG or GIF techniques now known in the art or
other techniques yet to be developed. Other color spaces may also
be used, as desired by the skilled artisan, particularly when
detection of a sought-after characteristic is enhanced thereby.
[0185] The present invention has now been described in connection
with a number of particular embodiments thereof. Additional
variations should now be evident to those skilled in the art, and
are contemplated as falling within the scope of the invention.
[0186] All references cited herein are incorporated herein by
reference in their entirety.
* * * * *