| Method for detecting infectious agents using computer controlled automated image analysis -> Monitor Keywords |
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Method for detecting infectious agents using computer controlled automated image analysisRelated Patent Categories: Image Analysis, Applications, Dna Or Rna Pattern Reading, Cell Analysis, Classification, Or CountingMethod for detecting infectious agents using computer controlled automated image analysis description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20060239533, Method for detecting infectious agents using computer controlled automated image analysis. Brief Patent Description - Full Patent Description - Patent Application Claims
[0001] This application claims priority benefits of U.S. Patent Application No. 60/295,587 filed Jun. 4, 2001, the entire disclosure of which is incorporated herein by reference. 1. 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". 2. 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; Halne, 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. Vasc. 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. 3. SUMMARY OF THE INVENTION [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, e.g., an 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 further 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. Continue reading about Method for detecting infectious agents using computer controlled automated image analysis... Full patent description for Method for detecting infectious agents using computer controlled automated image analysis Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Method for detecting infectious agents using computer controlled automated image analysis patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. 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