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    • 专利摘要:
    METHOD AND SYSTEM OF DETECTING A PATHOGEN INFECTION IN A SAMPLE. This document discloses, among other things, a method, kit and systems for detecting a pathogen infection in a body sample the method comprising (i) staining said body sample with one or more dyes, comprising at least one predominant dye staining DNA to thus provide differential staining between DNA and at least one other cellular component being different from DNA; (ii) the identification of at least one colored area comprising the DNA, if it exists in the sample, and at least one other colored area comprising the other cellular component; (iii) the extraction of structural features for the first stained area and at least one other stained area, said structural features comprise at least one of (i) the size of at least one of the first stained area and another stained area and ( ii) location of said first colored area and said at least one other colored area, one in relation to the other; and (iv) determining the presence of a suspected pathogen in the body sample if a first stained area has been identified and said structural features conform to predetermined structural features such as (...).
    公开号:BR112014016072B1
    申请号:R112014016072-4
    申请日:2012-12-27
    公开日:2021-01-12
    发明作者:Joseph Joel Pollak；Daniel Levner；Yonatan Bilu；Arnon Yafin；Noam Yorav-Raphael；Yuval Greenfield
    申请人:Sight Diagnostics Ltd.；
    IPC主号:
    专利说明:

    [0001] [001] The present disclosure relates to diagnostic methods and in particular, methods and systems for detecting pathogens in body fluids. PREVIOUS TECHNIQUE
    [0002] [002] References considered relevant as historical to the material disclosed here are listed below:
    [0003] [003] J. Keizer, J. Utzinger, Z. Premji, Y. Yamagata and B. H. Singer in Annals of Tropical Medicine & Parasitology, Vol. 96, No. 7, 643-654 (2002)
    [0004] [004] US Patent No. 5,470,751
    [0005] [005] Online publication of the Centers for Disease Control and Prevention CDC, Diagnostic Procedures, 2009 "Blood specimens: Microscopic Examination" at http://www.dpd.cdc.gov/dpdx/HTML/Frames/DiagnosticPro cedures / body_dp_bloodexamin .htm
    [0006] [006] The recognition of the references above in this document should not be interpreted to mean that they are in any way relevant to the patentability of the material disclosed here. HISTORIC
    [0007] [007] Staining is a technique often used in biology and medicine to highlight structures in biological tissues for visualization, often with the help of different microscopes. Stains can be used to define and examine bulky tissues (highlighting, for example, muscle fibers or connective tissue), cell populations (classifying different blood cells, for example), or organelles within individual cells.
    [0008] [008] Cell staining is used to better visualize cells and cellular components under a microscope. When using different stains, it is preferable to stain certain cellular components, such as a nucleus or a cell wall, or the entire cell. The stains can be used in fixed or non-living cells, as well as in living cells.
    [0009] [009] Cell staining techniques are also often used to detect a pathogen in a blood sample. An important pathogen is malaria.
    [0010] [010] The first dye synthesized for stain detection was methylene blue in 1876. Six years later, it was used to discover tuberculosis bacilli. Over the years, modifications of MB have been employed, including "cured" MB in combination with other dyes, such as eosin, which have allowed differentiation between blood cells and the nuclei of malaria parasites, as well as the combination of demethylation of blue of MB methylene with glycerol as a stabilizing agent in methanol solvent, known as the Malacowski-Wright-Giemsa dye.
    [0011] [011] J. Keizer et al. describes Acridine Orange in comparison to Giemsa's dye, for malaria diagnosis, including its diagnostic performance, its promotion and implementation and the implications for malaria control.
    [0012] [012] US Patent No. 5,470,751 describes another reagent for staining cells infected with malaria and a method for detecting cells infected with malaria using the same, wherein the reagent is a staining solution comprising at least a first dye of an analog of Auramine, such as Auramine O and at least a second dye of a condensed benzene derivative, such as 3,3'-diethyl-2,2'-oxacarbocyanine iodide. A test sample is treated with a reagent to stain cells infected with malaria. Stained malaria-infected cells are then detected optically.
    [0013] [013] In addition, the Centers for Disease Control and Prevention CDC, Diagnostic Procedures, 2009 describes under the title "Blood specimens: Microscopic Examination" that Kawamoto's technique for detecting blood parasites, such as malaria parasites, using fluorescent dyes that color nucleic acids. As discussed in this publication, in the Kawamoto technique, blood smears on a slide are stained with acridine orange (AO) and examined with a fluorescence microscope or a light microscope adapted with an interference filter system. This results in a differential staining of nuclear DNA in green and cytoplasmic RNA in red, which allows the recognition of parasites. GENERAL DESCRIPTION
    [0014] [014] The present disclosure provides, according to a first of its aspects, a method of detecting a pathogen infection in a sample, the method comprising:
    [0015] [015] - the staining of said body sample with one or more dyes, comprising at least one predominant dye staining DNA, thus providing differential staining between DNA and at least one other cellular component being different from DNA;
    [0016] [016] - the identification of at least one stained area comprising the DNA, if it exists in the sample, and at least one other stained area comprising the other cellular component;
    [0017] [017] - the extraction of structural characteristics for the first colored area and at least one other colored area, said structural characteristics comprise at least one among (i) the size of at least one among the first colored area and another colored area and (ii) the location of said first colored area and said at least one other colored area, one in relation to the other; and
    [0018] [018] - the determination of the presence of a suspected pathogen in the sample if a first stained area has been identified and said structural features conform to predetermined structural characteristics as characterizing the suspected pathogen.
    [0019] [019] According to a second aspect, the present disclosure provides a kit comprising:
    [0020] [020] - a first dye predominantly staining DNA;
    [0021] [021] - a second dye to stain at least one other cellular component being different from DNA;
    [0022] [022] - instructions for using the first dye and said second dye to determine the presence of a suspected pathogen in a sample. According to a third aspect, the present disclosure provides a system comprising:
    [0023] [023] - an image capture component configured and operable to acquire at least one optical image of a colored area in a sample, the colored area comprising a first colored area corresponding predominantly to DNA and at least one other colored area corresponding to at least at least one other cellular component being different from DNA;
    [0024] [024] - an image processing unit configured for
    [0025] [025] i) extracting structural features from at least said first stained area and said other stained area, said structural features comprise at least one of (i) the size of at least one of said first stained area, and (ii) another colored area; and a location of said first stained area and said other stained area, in relation to each other; and
    [0026] [026] ii) determine the presence of a suspected pathogen in the body sample, if a first stained area is identified and said structural features conform to predetermined structural features as characterizing a pathogen infection.
    [0027] [027] In addition, according to a fourth aspect, the present disclosure provides a processing unit for identifying a pathogen in a sample, comprising:
    [0028] [028] - input module configured and operable to receive data corresponding to a stained object in the sample comprising at least one first stained area corresponding predominantly to DNA and at least one other stained area corresponding to at least one other cellular component other than DNA, and
    [0029] [029] - an image processing unit configured and operable to process said data corresponding to said colored object, the processing comprises:
    [0030] [030] - the extraction of structural features for at least the first stained area and the second stained area on the stained object, said structural features comprise at least one of (i) the area of at least one of the first and the other stained area and (ii) a location of said first stained area and said other stained area, in relation to each other; and
    [0031] [031] - the provision of a value or combination of values being indicators of the presence or absence of a suspected pathogen in the sample, if said structural characteristics conform to predetermined structural characteristics as characterizing the suspected pathogen
    [0032] [032] According to a fifth aspect, the present disclosure provides a computer program product comprising a computer-usable medium having computer-readable program code incorporated in it to detect a pathogen infection in a sample being stained with two or more dyes, the computer program product comprising:
    [0033] [033] computer-readable program code to cause the computer to identify in the stained object at least one first stained area corresponding predominantly to DNA and at least one other stained area corresponding to at least one other cellular component other than DNA;
    [0034] [034] computer-readable program code to cause the computer to extract structural features for at least the first stained area and another stained area, said structural features comprise at least one of (i) an area of at least one of the first and another colored area and (ii) the location of said first colored area and said other colored area, in relation to each other;
    [0035] [035] computer-readable program code to cause the computer to determine a value or combination of values being indicators of the presence of a suspected pathogen in the sample, if said structural features conform to predetermined structural features as characterizing the infection of suspected pathogen. BRIEF DESCRIPTION OF THE DRAWINGS
    [0036] [036] To better understand the material that is revealed in this document and to exemplify how it can be carried out in practice, achievements will now be described, just as a non-limiting example, with reference to the attached drawings, in which:
    [0037] [037] Figures 1A to 1F show images obtained in three different exposures; images of bright field (Figures 1A, 1D), blue fluorescence (365nm, Figures 1B and 1E) and UV fluorescence (475nm, Figures 1C, 1F) of white blood cells (Figures 1A-1C) and red blood cells infected with malaria (Figures 1D-1F). DETAILED DESCRIPTION OF ACHIEVEMENTS
    [0038] [038] The present invention is based on the development of methods for differential staining of different components in a blood sample to thus allow an almost instantaneous detection of an infection in the blood.
    [0039] [039] Specifically, it was discovered by the inventors that staining a blood sample with an acridine orange reagent (AO) allows differentiation between pathogens containing DNA and, for example, platelets, reticulocytes, Howell-Jolly bodies, and bacteria such as Staphylococcus epidermidis.
    [0040] [040] It was further discovered that staining a blood sample with AO and Hoechst reagents and employing an image analysis of the stained object including the two different stained areas (the object being an image in the field of view comprising the stained areas under analysis), allowed the detection of malaria infection in the blood sample. In this connection, the inventors found that, in addition to detecting two different colored areas in the visualized colored object (of the sample), it is also sometimes important to take into account some spatial characteristics of the colored areas, such as the size or dimensions of each colored area and / or the spatial relationship between them.
    [0041] [041] The inventors also determined that, to detect a pathogen in the blood sample, at least one dye must stain deoxyribonucleic nucleic acid (DNA) with preference over staining other cellular components.
    [0042] [042] It was also discovered by the inventors that the diagnostic sensitivity obtained using two dyes, one predominantly staining DNA, is above 95%, and even above 97% or 99%.
    [0043] [043] In addition, it was discovered, and it was also demonstrated in the second example, that the method revealed in this document allows to identify the presence of a pathogen at low counts / μl. For example, the example provided in this document shows that a parasite, such as Plasmodium, can be detected in the blood even at a count below 1,000 parasites per μl and even at a low count of about 20 parasites per μl
    [0044] [044] Thus, according to a first aspect, the present disclosure provides a method of detecting a pathogen infection in a sample, the method comprising:
    [0045] [045] - the staining of said body sample with one or more dyes, with at least one predominant dye staining DNA, thus providing differential staining between DNA and at least one other cellular component being different from DNA;
    [0046] [046] - the identification of at least one stained area comprising the DNA, if it exists in the sample, and at least one other stained area comprising the other cellular component;
    [0047] [047] - the extraction of spatial characteristics for the first colored area and at least one other colored area, said spatial characteristics comprise at least one among: (i) size of at least one among the first colored area and another colored area ; and (ii) location of the first stained area and at least one other stained area, in relation to each other;
    [0048] [048] - the determination of the presence of a pathogen in the sample if a first stained area has been identified and said spatial characteristics conform to (namely, correspond to, fit into, fall within, are associated with), a value or a predetermined combination of values characterizing a suspected pathogen infection.
    [0049] [049] As appreciated, although the invention is described in the following detailed description with reference to a method of detecting a pathogen infection in a sample, such as a blood sample, it should be understood that the present disclosure also encompasses a kit to perform the invention, a system, a processing unit and other aspects, as disclosed in this document,
    [0050] [050] The method of the invention is applicable to a variety of samples. In some embodiments, the sample is a body sample. In some embodiments, the body sample is a sample of body fluid, such as, among others, blood, salive, semen, sweat, sputum, vaginal fluid, feces, breast milk, bronchoalveolar lavage, gastric lavage, tears and nasal discharge.
    [0051] [051] In some embodiments, the body sample is a blood sample.
    [0052] [052] In some embodiments, the blood sample is selected from a whole blood sample, a red blood cell sample, a leukocyte cream sample, a plasma sample, a serum sample, a sample of any other blood fraction, or any combination of themselves.
    [0053] [053] The body sample can be from any living creature, but preferably from warm-blooded animals.
    [0054] [054] In some embodiments, the body sample is a sample of a mammal.
    [0055] [055] In some embodiments, the body sample is obtained from a human body.
    [0056] [056] In some other embodiments, the sample is collected from any domestic animal, zoo animals and farm animals, including, but not limited to, dogs, cats, horses, cows and sheep.
    [0057] [057] In some embodiments, the body sample can be collected from animals that act as disease vectors including deer or rats.
    [0058] [058] In some other embodiments, the sample is an environmental sample, such as, among others, water sample (for example, groundwater), surface swab, soil sample, air sample, or any combination thereof.
    [0059] [059] In some embodiments, the sample is a food sample, such as, among others, meat sample, dairy product sample, water sample, washing liquid sample, beverage sample, and any combination thereof.
    [0060] [060] As a first stage in the method disclosed in this document, the sample is stained with at least two dyes providing, under suitable conditions, two distinct stained areas in the sample.
    [0061] [061] A colored area can be defined, among other ways, in relation to, for example, background or collateral coloring. For example, an intensity-based threshold, contrast-based threshold, edge detection, background detection, normalization, or a combination of these can be applied to define the colored area. In addition, the definition of the colored area may allow, among others, fuzzy borders, for example, using a brightness-weighted sum / integral.
    [0062] [062] In some embodiments, such as in the case of membrane staining, a first stained area residing within a second stained area can be defined including a case in which the first area resides within the area enclosed within the second area. For example, red blood cell membrane staining may result in a second stained area that resembles narrow lines around the cell's circumference (as seen in a two-dimensional microscope image); in such a case, it can be determined that the first area resides within said second area if it is framed within the circumference of the stained cell.
    [0063] [063] In some embodiments, standard microscopic sample preparation may be required to stain the sample. For example, sample preparation can take advantage of the well-known method of thin smear or thick smear for blood smear preparation. As an alternative example, a drop of the sample is placed in the middle of a clean slide along with, before or after placing the dye and a cover slip is gently placed over the drop at an angle, with an edge touching the cover slip first. The liquid is then allowed to spread between the two pieces of glass without applying pressure.
    [0064] [064] When referring to a dye, it is understood that it encompasses any chemical or biological substance that is capable of staining a component of a biological cell, to improve the contrast and highlight structures of the stained object, be it a cell or part of a cell. The dye may have a preference or class specificity, for example, it may have preference or specificity for staining nucleic acids and among the nucleic acid, for DNA or RNA, preference for amino acids, lipids, carbohydrates, etc.
    [0065] [065] When referring to the predominant preference or coloring, it should be understood that the dye colors another cellular component in a specific color or fluorescence that is at least twice, three times, four times or even 10 times greater in intensity than its staining intensity of another cellular component in the same color or fluorescence spectrum.
    [0066] [066] In some embodiments, when referring to the predominant preference or coloring, it should be understood that the dye has an affinity (molecular attraction) to a cellular component (in the specific color or fluorescence spectrum) that is at least twice, three times , four times or even 10 times greater in its affinity to another cellular component (in the same color or fluorescence spectrum).
    [0067] [067] In some additional realizations, when referring to the predominant preference or staining, it should be understood that the staining of DNA by the dye is stable or has more stability compared to its staining of other components. Stability can be understood as the coloring produced by the dye remains substantially consistent for at least 30 minutes after being brought into contact with the dye, sometimes at least 1 hour, 2 hours or even 5 hours after staining the sample with the dye having preference to DNA. Alternatively, stability can be understood as the coloration produced by the dye remains substantially consistent during exposure to light (for example, light used for fluorescence excitation) for at least 0.25 seconds, 1 second or even 15 seconds of exposure.
    [0068] [068] In this context, it should be understood that the dye having preference to DNA can also stain other cellular components but with less attraction or less intensity or with a different fluorescence response (excitation spectrum and / or emission spectrum) so that allows greater improvement of DNA, whose dye is preferred. For example, as will be further discussed below, under some conditions, a dye can predominantly stain DNA, however, under some other conditions, the same dye can stain RNA.
    [0069] [069] Likewise, the staining of another cellular component must be understood by encompassing the staining of one or more cellular components that are DNA. This can also include staining one or more cellular components in addition to DNA, but with no preference or less preference for staining of DNA.
    [0070] [070] In some embodiments, the dyes are not specific to the cell type. In other words, the dye is not specific to a specific pathogen or to a specific stage in the life cycle of a specific pathogen or to a specific cell in the host being infected with it and will stain a cell component regardless of its origin, for example, a DNA sequence or structure itself, an RNA sequence or structure itself, protein itself, etc.
    [0071] [071] The existence of a first stained area is determined based on several factors that may refer, among others, to the intensity of the color, the shape of the color, the variability or consistency of the color, etc. Once the existence of a first stained area is determined, it is attributed to the presence of DNA.
    [0072] [072] The method also provides a stained area of at least one cell component other than DNA. The stained area can be obtained by the same dye, under different conditions or by using a different dye.
    [0073] [073] Without being limited to them, the at least one other cellular component is selected from the group consisting of RNA, proteins, lipids, membrane, cytoplasm, ribosomes, carbohydrates, glucans, glycoproteins, endoplasmic reticles, or any combination thereof.
    [0074] [074] In some embodiments, the at least one other cellular component is RNA.
    [0075] [075] There are a variety of dyes that can be used according to the present disclosure. In some embodiments, the dye is a chromophore or fluorophore.
    [0076] [076] Dyes such as Giemsa staining are known as chromogenic - their effect is to provide color or opacity to the sample and are visible, for example, under bright field microscopy.
    [0077] [077] In some embodiments, the dye provides fluorescent staining of the sample. Fluorescence is visualized by illuminating the sample with an "excitation" light spectrum, which results in an "emission" in a different light spectrum. Among the potential advantages of fluorescent stains is the fact that the regions of the sample that are not stained show as dark or almost dark, thus typically providing greater contrast between stained and unstained areas than chromogenic stains. Acridine Orange (AO) is an example of a dye used to stain biological samples with fluorescence.
    [0078] [078] In some embodiments, the dye is a cell-permeable dye.
    [0079] [079] Without being limited to them, a dye that predominantly colors DNA can be any member of the group consisting of acridine orange (AO, N, N, N ', N'-Tetramethylacridine-3,6-diamine, green coloring ), Hoechst family, DAPI (4 ', 6-diamidino-2-phenylindol), ethidium bromide (3,8-Diamino-5-ethyl-6-phenylphenanthridinium bromide), propidium iodide (2.7-iodide methyliodide -Diamino-9-phenyl-10 (diethylaminopropyl) -phenanthride), SYBR family, YOYO, DRAQ family, SYTO family, TOTO family, or any combination thereof. In addition, the dye comprises a chemical modification of any of the aforementioned dyes that preserves your preference for DNA.
    [0080] [080] Without being limited to them, a dye that does not predominantly color DNA can be any member of the group consisting of crystal violet (Tris (4- (dimethylamino) phenyl) methyl chloride), hematoxylin stains, eosin stains, Safranin (symmetrical 2,8-dimethyl-3,7-diamino-phenazine azonium compounds), acridine orange (AO, N, N, N ', N'-Tetramethylacridine-3,6-diamine, red staining), acid-Schiff stains, Masson stain, Prussian blue, or any component or combination thereof. Many more stains that do not predominantly color DNA are known in the art.
    [0081] [081] Dyes such as AO provide different fluorescence spectra for different cellular components. When AO colors DNA at neutral pH, it has a maximum excitation at 502 nm (cyan) and a maximum emission at 525 nm (green); when it colors RNA at neutral pH, the maximum excitation changes to 460 nm (blue) and the maximum emission changes to 650 nm (red). As such, it allows for differential staining between DNA and RNA, depending on the excitation wavelength and sample conditions.
    [0082] [082] The Hoechst family of dyes is known for the chemical formula C25H26N6R, with R representing a variety of possible substituents, such as, among others, OH (Hoechst 33258); -CH2CH3 (Hoechst 33342), -N (CH2) 2 (Hoechst 34580), -SO2NH2 (Hoechst S769121).
    [0083] [083] In some embodiments, the method involves staining combined with at least two dyes, the first dye being a member of the Hoechst family and the second dye being acridine orange.
    [0084] [084] When referring to a combination of two or more stains, it should be appreciated that the two or more stains can be added to the sample simultaneously or in sequence. Likewise, the respective stained areas can be obtained simultaneously or in sequence, as long as there is a point in time that allows visualization of two or more areas stained differently in the same sample.
    [0085] [085] In one embodiment, the method employs a combination of AO with the membrane permeable dye that is essentially specific to nuclear DNA (for example, Hoechst 33342, yielding blue emission).
    [0086] [086] In accordance with this realization, AO allows the use of red emission or a combination of red and green emissions to stain the pathogen's cytoplasm (and possibly the nucleus, but without substantially interfering with Hoechst staining).
    [0087] [087] In some embodiments, the combination of AO and Hoechst allows the detection of a parasite. According to this realization and without being limited to it, for the detection of a parasite, such as a member of the Plasmodium species, the concentration of Hoechst's reagent, such as Hoechst 33342, can be 10µg / mL, but other possible values may also be used, for example, in the range of 3µg / mL to 250µg / mL; and AO can be used at 1.5 µg / mL, but other possible values can also be used, for example, in the range between 0.2 µg / mL and 125 µg / mL.
    [0088] [088] In some embodiments, the amount of AO and Hoechst's reagent to be used is determined in order to provide, in the biological sample, a proportion between them. Therefore, the Hoechst: AO ratio can be in the range between 50: 1 and 1: 1, sometimes around 7.5: 1.
    [0089] [089] The same proportion, or similar, may also be applicable for other dye combinations, a first dye predominantly staining DNA and the other dye, another component, as defined, namely, a relationship between the first dye and another dye in the range between 50: 1 and 1: 1.
    [0090] [090] AO can, in addition, or alternatively, be used to stain, for example, Plasmodium cytoplasm and / or food vacuole, but not the red blood cell (RBC) cytoplasm within which the parasite may be, allowing the parasite's body to be seen without being obscured by an RBC, even if present within the RBC.
    [0091] [091] Appropriate concentrations can be optimized for factors such as the specific dye combination, desired duration of the color incubation, resulting color brightness or fluorescence, and resulting color character.
    [0092] [092] In some embodiments, the method disclosed in this document is applicable for the detection of an infection by a pathogen carrying DNA. As such, at least the first dye colors at least the DNA, if present in the sample, to thus provide a first stained area indicative of the presence of the pathogen carrying DNA in the sample.
    [0093] [093] The pathogen can be an infectious microorganism. In some embodiments, the pathogen is a eukaryotic parasite. When referring to the eukaryotic parasite, in the context of the present disclosure, it should be understood to include unicellular and multicellular parasites, but also fungi, such as yeast (eg Candida) and Aspergillus.
    [0094] [094] In some embodiments, pathogens are bacteria. This includes, for example, and is not limited to, Escherichia coli, Staphylococcus aureus, Microbacterium tuberculosis, Salmonella species, Borrelia species and Treponema pallidum and others known.
    [0095] [095] In some embodiments, the pathogen is a eukaryotic parasite. According to this embodiment, the parasite can be a unicellular parasite, such as protozoa. This includes genital protozoa, for example, Trichomonas vaginalis, protozoa of the nervous system, for example, Naegleria fowleri, fecal protozoa, for example, Giardia lamblia, blood protozoa. In some embodiments, the pathogen can be a multicellular parasite, such as Wuchereria bancrofti, Brugia malayi, Brugia timori, Mansonella streptocerca, or Onchocerca volvulus.
    [0096] [096] In some embodiments, the parasite is a protozoan selected from the genera consisting of Trypanosoma (which causes Chagas' disease and African sleeping sickness); Plasmodium (which causes Malaria); Toxoplasma (causing Toxoplasmosis); Babesia (cause of Babesiosis).
    [0097] [097] Specifically, when referring to Plasmodium, it should be understood encompassing at least any member of the group consisting of Plasmodium falciparum (P. falciparum), Plasmodium vivax (P. vivax), Plasmodium ovale (P. ovale), Plasmodium malariae (P. malariae), and Plasmodium knowlesi (P.knowlesi).
    [0098] [098] In some embodiments, the pathogen is understood to mean a specific stage in the life cycle of a specific pathogen or group of it. For example, the invention disclosed in this document can be applied specifically to the detection of trophozoites, schizonts or gametocytes of the species of Plasmodium or P. falciparum in particular.
    [0099] [099] As can be appreciated, the method disclosed in this document can be applicable for the detection of multiple pathogens using the same conditions and / or in the same sample, for example, the same combination of dyes, the same test conditions, etc., as well as for the detection of a pathogen in multiple stages of its life cycle. In some embodiments, the method disclosed in this document may determine which of the one or more of the multiple pathogens (or stages of life) is the suspect.
    [0100] [0100] In some embodiments, the method disclosed in this document is to detect Plasmodium infection in a human blood sample, the method comprising:
    [0101] [0101] - staining of the human blood sample with at least two dyes, under conditions that allow at least the staining of DNA and at least one other cellular component being different from DNA;
    [0102] [0102] - the identification of at least one stained area comprising DNA, if it exists in the blood sample, and at least one other stained area comprising another cellular component in the blood sample;
    [0103] [0103] - the extraction of structural characteristics for the first colored area and at least one other colored area, said spatial characteristics comprising the size of at least one among the first colored area and another colored area and a location of the first colored area and at least one other area colored in relation to the other;
    [0104] [0104] - determination of value or combination of values being indicators of the presence of suspicious Plasmodium in the blood if a first stained area has been identified and said structural characteristics fall within predetermined limitations as characterizing a pathogen infection.
    [0105] [0105] In some embodiments regarding the determination of the presence of Plasmodium in the blood, the stained object is obtained by staining the blood sample with a combination of acridine orange (AO) and Hoechst's dye, in particular, Hoechst 33342 with a proportion concentration between AO and Hoechst 33342 in the 1:50 to 1: 1 range, sometimes even in the 1:75 range.
    [0106] [0106] According to the above embodiment, the method is applicable for detecting malaria infection in a human blood sample.
    [0107] [0107] Once the sample is stained with one or more dyes, at least one stained object is obtained and the method then involves selecting at least one stained object in the sample and obtaining structural or spatial characteristics of the stained object.
    [0108] [0108] When referring to the spatial or structural characteristics of a colored object, it should be understood to include any measurable characteristic of the colored areas in the selected colored object. The characteristics can be presented as a discrete value or a range of values characterizing the colored area.
    [0109] [0109] In some embodiments, structural features include at least one of the size (for example, dimension, length, circumference, minimum width, maximum width or area) of the colored areas and / or the location of the first colored area in relation to the location of the other area or colored areas (s). In other words, the method can be carried out based on at least the determination of the dimensions, for example, the area of the colored areas, or the location of the first colored area in relation to the location of the other colored area (s). These features can also be combined with one or more additional features.
    [0110] [0110] Additional structural features may include, but are not limited to, shape of the stained area (s), movement pattern of the stained area (s), intensity of the area (s) ( s) stained, color of the stained area, texture (outline) of the stained area, sharpness of the stained area border, staining pattern in intensity or color, overlapping of the stained area with other stained areas or image characteristics, relative size from the colored area to other colored areas or image characteristics, proximity or contact of the colored area to other colored areas or image characteristics
    [0111] [0111] In some embodiments, the body sample is a blood sample.
    [0112] [0112] In some embodiments, the pathogen is a blood parasite.
    [0113] [0113] In some embodiments, the body sample is human blood and the pathogen is Plasmodium species responsible for human malaria, as are those selected from the group consisting of P. falciparum, P. vivax, P. ovale and P. malariae.
    [0114] [0114] When the body sample is a blood sample, the extraction of structural features comprises the selection of a stained object in the blood sample that comprises the first stained area and at least one other stained area.
    [0115] [0115] Based on the stained areas and the analysis of structural characteristics, a high probability of the sample being infected with a suspected pathogen is determined. Thus, in the context of the present disclosure, when referring to the determination of the presence of a suspected pathogen or pathogen, it should be understood that any of the following is determined:
    [0116] [0116] that one or more suspected pathogens are present in the sample;
    [0117] [0117] that one or more suspected pathogens are present in the sample at confidence levels above a predefined threshold; confidence levels may increase or a final determination of the presence of a suspected pathogen may require one or more additional analysis steps.
    [0118] [0118] that one or more suspected pathogens may be present in the sample with determined or estimated confidence scores or probabilities that are available for further analysis steps
    [0119] [0119] According to some realizations, it is determined that a suspicious pathogen is present in the sample if a first stained area is identified (being indicative of the presence of DNA in the sample), and the first stained area borders, is in proximity to , or resides within another stained area and the size of at least one of the first stain, that is, the area containing DNA, and the other stain is within a predetermined range. For example, an area containing DNA often resides next to an area stained for RNA, which exists in the cytoplasm.
    [0120] [0120] According to some other realizations, it is determined that the suspected pathogen is present in the sample if a first stained area is identified (again, being indicative of the presence of DNA in the sample) and the size of the first stained area satisfies a predetermined relationship in relation to the size of a colored area. This criterion can be used, for example, to differentiate eukaryotic parasites from bacteria: in eukaryotic cells, the area predominantly stained for DNA should be smaller than a cytoplasmic stain, indicating a distinct nucleus that is not present in bacteria.
    [0121] [0121] According to some other realizations, the presence of a pathogen is determined if a first stained area is identified and the dimensions or area of the first stained area is within a range of predetermined values to be associated with the specific pathogen or with a specific stage in the life cycle of a specific pathogen. For example, if the pathogen was the cause of malaria (ie Plasmodium), the area of the first stained area would typically not be below 0.2m or above 20pm or would be in the range of 0.2µm2 to 20µm2. In some realizations, the area of the first stained area in the detection of Plasmodium would not be below 0.8µm2 or above 13µm2 or would be within the range of 0.8µm2 to 13µm2.
    [0122] [0122] According to some other realizations, the presence of a pathogen is determined in the sample if a first stained area is identified and the areas of another stained area are within a range of predetermined values to be associated with the specific pathogen or a specific stage in the life cycle of a specific pathogen. For example, if the pathogen was the cause of malaria (ie Plasmodium), the area, for example, a cytoplasm, from the other stained area would typically not be below 0.8µm2 or above 65µm2 or would be in the range of 0, 8µm2 to 65µm2. In some cases to detect malaria infection, the area of the other colored area is not below 1.6µm2 or does not exceed 40µm2 or is in the range of 1.6µm2 to 40µm2.
    [0123] [0123] Considering the above characteristics, it is understood that, according to some realizations of the present disclosure, the specific area of the first colored area cannot exceed the specific area of the other colored area. Likewise, the dimensions of the first colored area should be statistically significantly (for example, by t-test) smaller than the dimensions of the other colored area.
    [0124] [0124] In some other embodiments, the presence of a suspect pathogen is determined in the sample if a first stained area is identified and the first stained area occupies a predetermined volume% of all other stained areas. For example, when referring to malaria, the first stained area would typically occupy between 12% and 60% of said stained area.
    [0125] [0125] According to some other realizations, the presence of a suspected pathogen is determined in the sample if a first stained area is identified and the first stained area and another stained area have predetermined shapes. For example, the Trypanosoma brucei triptomastigote stage is known to have an elongated, axis-like shape. Other shapes may include, but are not limited to, spiral, oval, elongated and spherical properties.
    [0126] [0126] According to some realizations, the presence of a suspicious pathogen is determined in the sample if a first stained area is identified and the first stained area has a variability in intensities that is above a predetermined threshold. Intensity variability can appear as a grouping of more than one area stained with the first stain (plurality of the first stained area) or as a first irregularly shaped stained area having different shades of color or fluorescence. This may reflect, for example, a mononucleated stage in the development of a protozoan, such as Plasmodium schizonts.
    [0127] [0127] Furthermore, according to some realizations, the presence of a suspicious pathogen is determined if a first stained area is identified and the pattern of directionality and / or speed of movement) of the stained object corresponds to a predetermined pattern being associated with the suspected pathogen. For example, when considering Trypanosoma brucei, its movement (mobility) is expected to occur in a characteristic pattern. As such, when the sample is stained with AO and Hoechst staining, the movement of the AO stained area can be analyzed to confirm an expected match or correlation with the expected pattern.
    [0128] [0128] In yet other embodiments, the presence of a suspected pathogen is determined if, during the movement of the colored object, the first colored area is kept within the limits of the other colored area. Thus, when referring to the example above with Trypanosoma brucei, the movement of the area stained with Hoechst can be analyzed to ensure that a supposed nucleus remains at all times within the area stained with mobile AO.
    [0129] [0129] The structural characteristics can be determined simply by viewing the sample, however, in some embodiments, they are preferably determined based on one or more optical images of the sample. Thus, according to some realizations, the method comprises capturing at least one optical image of the colored object and extracting the structural characteristics of at least one optical image.
    [0130] [0130] Sometimes, particularly, when the structural parameter includes the movement of the colored object, more than one optical image is captured and the structural parameter of the movement is determined based on a sequence of images over time.
    [0131] [0131] Needless to say, the more accumulated (or collected) the structural parameter (s) information of the stained object, the more accurate can be the determination and identification of a pathogen in the same sample.
    [0132] [0132] Structural characteristics can be analyzed manually, or by using a dedicated system to allow automated and / or relatively rapid detection and / or identification of the pathogen in the body sample. The analysis result can obtain a Yes / No indication of the presence of a pathogen, a probabilistic indication of the presence of a pathogen, images of a suspected pathogen for further analysis (manual or automatic) and / or can provide a more specific result, including the type of pathogen, level of invention (count / µl), etc.
    [0133] [0133] The use of an automated system can be of significance, not only to overcome human errors in the analysis of colored objects in the sample and to save time and labor, but also since certain components of a body sample may not stain immediately and / or the degree of staining can vary significantly over time, as is sometimes the case with Hoechst 33342 staining contaminated blood cells. Consequently, the characteristics of the image may vary depending on how fast the sample is analyzed. As such, there is an advantage to having an automated system allowing image acquisition and analysis of the stained sample quickly, or even almost instantaneously.
    [0134] [0134] Thus, according to an additional aspect of the present disclosure, a system is provided comprising:
    [0135] [0135] - an image capture component configured and operable to capture at least one optical image of a colored area in a sample, the colored area comprising a first colored area corresponding predominantly to DNA and at least one other colored area corresponding to at least at least one other cellular component being different from DNA;
    [0136] [0136] - an image processing unit configured to extract structural characteristics from said at least first colored area and said another colored area, said structural characteristics comprising at least the size of at least one among said first colored area, and another colored area; and a location of the first colored area and said other colored area, in relation to each other; and determining that a pathogen is present in the sample, if a first stained area is identified and the structural features conform to predetermined structural features as characterizing a pathogen infection.
    [0137] [0137] In some embodiments, the system also includes an output unit to provide an output comprising a value or a combination of values being indicative of the presence or absence of suspected pathogens in the sample.
    [0138] [0138] In some embodiments, the system is preferably computer-based using a set of parameters stored in it and corresponding to, among other things, structural characteristics associated with at least one defined pathogen. The parameters may, for example, include values or ranges of values expected from structural characteristics, associated with a pathogen, or parameters that control the execution of machine learning algorithms or data analysis. The parameters can be derived from information collected a priori or simultaneously obtained based on reference samples including predetermined pathogens.
    [0139] [0139] The parameters can also comprise information regarding the behavior of a pathogen, or cell infected with a pathogen, such as motility and variations in characteristics depending on the specific stage of the life cycle of a specific pathogen.
    [0140] [0140] The parameters can also comprise information regarding conditions to be used for the detection of a pathogen in a type of body sample, for example, in the blood sample, statistical information regarding the coloring qualities and variability of a set of dyes to be used for a specific body fluid, dye combinations or combinations of alternative conditions, etc.
    [0141] [0141] In some embodiments, the set of stored parameters comprises parameters associated with a plurality of pathogens. In some embodiments, the set of stored parameters comprises parameters that are associated with determining which of the one or more pathogens is the suspect.
    [0142] [0142] In some embodiments, the set of parameters is associated with pathogens transmitted by blood. In some other embodiments, the set of parameters is associated with blood parasites. In some specific embodiments, the set of parameters is associated with pathogens that harbor human blood, in particular, from a species of Plasmodium responsible for human malaria, such as those selected from the group consisting of P. falciparum, P. vivax, P ovale and P. malariae.
    [0143] [0143] An image processing unit for identifying a pathogen in a sample is also provided by the present disclosure, comprising:
    [0144] [0144] - input module configured and operable to receive (image) data corresponding to the stained object in the sample comprising at least one first stained area corresponding predominantly to DNA and at least one other stained area corresponding to at least one other cellular component other than DNA, and
    [0145] [0145] - an image processing unit configured and operable to process the data corresponding to the colored object, the processing comprises:
    [0146] [0146] - the extraction of structural features for at least the first stained area and another stained area on the stained object, said structural features comprising at least one among (i) the size of at least one among the first and the other areas stained and (ii) location of said first stained area and said other stained area, one in relation to the other; and
    [0147] [0147] - the provision of a value or combination of values being indicators of the presence or absence of a pathogen in the sample, if said structural characteristics conform to predetermined structural characteristics as characterizing a suspected pathogen.
    [0148] [0148] Several operating modes can be used in relation to the system and the image processing unit. Automated systems, as well as manual and semi-automated systems, can be employed.
    [0149] [0149] Regarding the system, the image capture component can be any device configured and operable to acquire at least an optical image of the colored object being selected, for example, a CCD or CMOS camera. The image capture component can be equipped with conventional features, such as focus features. The image capture component can also include lenses, aperture and shutter.
    [0150] [0150] The image capture component provides image data to be processed by the image processing unit. The image data in the context of image analysis is based on a set of pixel information that essentially covers one or more Regions of Interest (ROI).
    [0151] [0151] Image data typically including pixel information can be presented in any available format, including but not limited to Tagged Image File Format (TIFF), Joint Photographic Experts Group (JPEG), Graphic Image Format (GIF), Portable Network Graphics (PNG), Portable Document Format (PDF), bitmap (BMP), and raw image sensor data. The image data can be in a compressed data format or in an uncompressed data format; they can refer to any grayscale or color; can include bitmap data, or a portion of a bitmap file containing bitmap data.
    [0152] [0152] Once one or more images are captured, they are processed by the image processing unit. Processing by the processing unit is configured and operable to provide one or more values of structural characteristics of the colored areas. In other words, based on image analysis techniques, several output values referring to the structural characteristics of the stained areas are determined, which can allow the identification of the presence or absence of the suspected pathogen in the sample or an estimate of the probability of presence in the sample. Values may include the dimensions of one or more stained areas in the ROI, the relationship between the stained areas, and / or averages, composition or statistical values for structural characteristics. Processing may include, in some embodiments, the analysis of one or more values or a combination of values corresponding to structural characteristics in relation to the parameters (for example, values or ranges of values or appropriate machine learning weights) that have been determined a priori to characterize one or more pathogens, or differences between them.
    [0153] [0153] Image analysis can be based on one or more of the common practices, such as contour detection, image parameter derivatives, format detection, image reconstruction, segmentation, image differentiation, pattern matching, adapted filtering , machine learning and geometric hashing.
    [0154] [0154] The data processing by the image processing unit can use a classification module. The classification module can be provided with at least one structural parameter previously determined for a pathogen. The classification module can analyze the structural characteristics provided and deduce a selection of possible pathogens identified. As an illustration, a classifier can identify a plurality of possible pathogens or a single type of pathogen.
    [0155] [0155] In specific embodiments, the determined structural characteristics are associated with an expected pathogen and these are analyzed in relation to the predefined parameters.
    [0156] [0156] It should be noted that the identification of a single pathogen can be a complex process that includes different combinations of characteristics. Therefore, structural characteristics determined after the use of a specific dye combination can be used as criteria for further analysis of an additional dye combination. For example, structural features identified in a previous analysis can be used to continue the identification process while focusing on a subset of possible dye combinations. For example, at a biological family level of identification of a pathogen, the results of previous analyzes may provide a selection list of possible best filter (s) to be used in a next round of analysis.
    [0157] [0157] In some embodiments, image analysis and determination of the presence of suspicious pathogens or an indicative value or values with the presence of suspicious pathogens can take advantage of one or more machine learning algorithms, which operate on a plurality of structural characteristics or related values. Such algorithms include, among others, Support Vector Machines (SVM), artificial neural networks, Naive Bayes classifiers, Bayesian networks, decision trees, nearest neighbor algorithms, random forests, Boosting, regression analysis, linear classifiers, quadratic classifiers, nearest k-neighbors, hidden Markov models and any combination thereof. The result of one or more machine learning algorithms can comprise one or more of confidence scores, probabilities and / or yes / no decisions.
    [0158] [0158] As appreciated, the more characteristics determined in the analysis - the more accurate the identification process. By using a plurality of dyes to induce a plurality of structural characteristics, the risk of detection error or classification error is reduced. However, additional features can also correspond to a greater computational or operational load. Some achievements strive to balance the added benefit of additional features with the associated load drawbacks.
    [0159] [0159] Based on the value or combination of values obtained by the analysis, several decisions are made by the processing unit regarding the presence or absence of a pathogen in the sample, sometimes including the type of the pathogen (qualitative identification) and / or the quantity (quantitative identification) of the pathogen in the sample.
    [0160] [0160] The processing unit is also configured to communicate with an output unit. Thus, based on the output value or values of the structural characteristics determined by the processing unit, an output signal or output signals indicative of the presence or absence of at least one pathogen and, in some embodiments, the type of the pathogen, in the image acquired are provided by an output unit.
    [0161] [0161] The output can be provided in any acceptable form, including a graph, graphic or text displayed on a monitor of a control unit, a printout, such as a voice message, or a user's smartphone screen, to accept data processed from the processing unit and display information regarding the structural characteristics obtained and / or associated values determining the presence and optionally the identity of a pathogenic infection, using lists, tables, graphs, etc. A monitor can be connected to a printer to print the output.
    [0162] [0162] The processing unit may also include a user interface module, for example, a keyboard, or a touch screen to allow a technician to perform the method of some embodiments of the invention, to control the operation of the system, including , among other things, input data in relation to the source of the body fluid examined, date, location, etc.) system operating conditions, types of dyes used, number of images to be obtained, time interval between images, etc.
    [0163] [0163] Sometimes image analysis may also involve adjusting or normalizing the brightness of the image based on the degree of coloration of the sample. These can be based, for example, on the identification of one or more of the lightest and darkest pixel values in the image or set of images (for example, corresponding to a specific sample), average brightness of the lightest area and / or darker, and / or image histogram. Such characteristics can be extracted from a representative image (not necessarily the image being normalized) or from statistical analysis of multiple images. The characteristics used for normalization can be based on a single image or on multiple images, which can be captured using different excitation wavelengths (as in AO providing different colors under different illumination wavelengths).
    [0164] [0164] The brightness of the image can also be adjusted using other means of control, such as the component of image capture time of exposure and / or brightness of the illumination.
    [0165] [0165] In addition, the operating conditions of the system may allow the timing of image acquisition, for example, to allow sufficient incubation time with one or more dyes, as well as operation with different optical wavelength configurations. excitation and / or emission, to capture the image of the stained sample in different color or fluorescence spectra.
    [0166] [0166] To capture the image of the stained sample in different color or fluorescence spectra, changes in excitation can be achieved by changing the color of the lighting. This can be done, for example, by providing two or more light sources (for example, for AO, 365nm UV LED light and 475nm blue LED light) and combining them optically (for example, using a dichroic mirror or grid).
    [0167] [0167] In another example, a single light source (for example, 365nm UV LED light) can be used to excite two dyes simultaneously, and one or more optical filters are moved in or out of the optical path to select the relevant emission wavelengths. Other sets of dye can be simultaneously excited using the same incident illumination described here, even if one or more of the dyes are excited not optimally. As an example, AO can similarly be co-excited with "a Hoechst stain, DAPI and DRAQ stains.
    [0168] [0168] In yet another example, a single light source (for example, 365nm UV LED light) can be used to excite two or more dyes simultaneously, and the emission optical path is divided so that the two or more Emissions are captured in two or more image capture components.
    [0169] [0169] In yet another example, a color image acquisition sensor is used to simultaneously capture two or more fluorescence signals. The use of an image acquisition sensor can, for example, avoid the need for one or more optical filters that are moved in or out of the optical path to select the relevant wavelength.
    [0170] [0170] In the context of the present disclosure, several sources of illumination can be used, these include, among others, those that come from white light (as in bright light microscopy), UV light, blue light, green light, yellow light, light red, combinations thereof, or any light applicable to excite one or more of the dyes used for coloring.
    [0171] [0171] The system components, namely, the image capture component, the processing unit, the output unit, etc., can be connected directly to each other (for example, directly by a wire) or one or more of the components may be remote from one or more other components. For example, the image capture device can send data to a processing unit over an intranet or over the internet, to allow processing at a remote location.
    [0172] [0172] An example of a system that can be used to carry out the method of the present disclosure is described in PCT patent application publication No. WO 2012/090198, the content of which is incorporated in this document, in its entirety, by reference .
    [0173] [0173] The processing unit is typically operable by executing a dedicated instruction program (for example, software application) that performs the analysis and storage of input data. The software application can be embedded in a machine-readable program storage device, such as a CD or a memory disk.
    [0174] [0174] In accordance with the above, the present invention provides a machine-readable program storage device, tangibly incorporating the machine-executable instruction program to perform a method of detecting a pathogen infection in a sample, the method comprising :
    [0175] [0175] - the staining of said fluid sample with two or more dyes, said two or more dyes providing differential staining of at least DNA and at least one other cellular component being different from DNA;
    [0176] [0176] - the identification of at least one first stained area corresponding predominantly to DNA and at least one other stained area corresponding to at least one other cellular component;
    [0177] [0177] - the extraction of structural features for at least one first stained area and the other stained area, said structural features comprising at least the size of at least one among the first and one other stained areas and a location of said first area stained and said another stained area in relation to the other;
    [0178] [0178] - the determination of a value or combination of values being indicators of the presence of a pathogen in the sample, if the said structural characteristics correspond to predetermined structural characteristics as characterizing a pathogen infection.
    [0179] [0179] In addition, the present disclosure provides a computer program product comprising a computer-usable medium having computer-readable program code incorporated in it to detect a pathogen infection in a body sample being stained with two or more dyes, the computer program product comprising:
    [0180] [0180] computer-readable program code to make the computer identify a colored object within the body sample;
    [0181] [0181] computer-readable program code to cause the computer to identify in the stained object at least one first stained area corresponding predominantly to DNA and at least one other stained area corresponding to at least one other cellular component other than DNA;
    [0182] [0182] computer-readable program code to cause the computer to extract structural features for at least one first stained area and another stained area, said structural features comprising at least the size of at least one of the first and another colored areas and a location of said first colored area and said another colored area in relation to each other;
    [0183] [0183] computer-readable program code to make the computer determine a value or combination of values being indicators of the presence of a suspected pathogen in the sample, if said structural characteristics correspond to predetermined structural characteristics as characterizing the pathogen infection .
    [0184] [0184] Finally, the present disclosure provides a kit comprising:
    [0185] [0185] - a first dye predominantly staining DNA;
    [0186] [0186] - a second dye to stain at least one other cellular component being different from DNA;
    [0187] [0187] - instructions for using the first dye and the second dye to determine the presence of a suspected pathogen in a body sample.
    [0188] [0188] According to this aspect, the first and second dyes can be provided in a single composition or in a first composition comprising the first dye and a second composition comprising the second dye. The first and second compositions each comprise, in addition to the dye, a vehicle suitable for use in coloring the body sample with the respective dyes.
    [0189] [0189] The kit revealed in this document must be used to perform each of the steps and conditions of the method revealed in this document.
    [0190] [0190] In some embodiments, the first dye comprises a Hoechst dye. In some embodiments, the second dye comprises acridine orange.
    [0191] [0191] In some embodiments, the kit includes instructions for the provision of the body sample with the first dye and the second dye in a ratio in the range between 50: 1 and 1: 1.
    [0192] [0192] In some embodiments, the first dye comprises Hoechst's dye, specifically Hoechst 33342 and the second dye comprises AO, the concentration of the first dye is in the range between 3µg / mL and 250µg / mL; and the AO concentration is in the range between 0.2 µg / mL and 125 µg / mL.
    [0193] [0193] In some other embodiments, the first dye comprises Hoechst dye, specifically Hoechst 33342 and the second dye comprises AO, the concentration of Hoechst 33342 is about 10µg / mL and the AO concentration is about 1.5µg / mL .
    [0194] [0194] In some embodiments, the first and second dyes can be combined with a suitable buffer, such as, among others, one comprising 1% Tris-EDTA buffer, 4.5% DDW and 92.5% saline.
    [0195] [0195] Without wishing to be linked to theory, it is possible that the AO / Hoechst staining proved more robust and stable staining, which allowed a fast and efficient machine view of the stained sample. This allows the acquisition of an image of a larger amount of blood than can be examined under a microscope in conventional methods, which, in turn, provided greater sensitivity in low parasitemia. It is also possible (in addition to, or alternatively) that one or more of the applied analysis criteria provided reliable results that are not dependent on the human factor.
    [0196] [0196] Some achievements of the present disclosure will now be exemplified in the following description of non-limiting examples that were carried out according to the disclosed method. It should be understood that these examples are intended to be in the nature of illustration rather than limitation. Obviously, many modifications and variations of these examples are possible in the light of the above teaching. Therefore, it should be understood that, within the scope of the appended claims, the invention can be practiced in another way, in a myriad of possible ways, than specifically described below. DESCRIPTION OF SOME NON-LIMITATIVE EXAMPLES DETECTION OF PLASMODIUM IN A BLOOD SAMPLE
    [0197] [0197] A blood sample obtained from a human subject being identified as having malaria infection was stained with a dye solution comprising 2µg / ml Acridine Orange, Hoechst 33342 15µg / ml, 1% Tris-EDTA buffer, DDW 4, 5% and saline 92.5%.
    [0198] [0198] After staining, images of the samples were obtained using a different lighting color, using 365nm UV LED light to scan the AO staining and blue LED light at 475nm, to scan the Hoechst staining). The sample was also digitized in the bright field (illuminated with white light).
    [0199] [0199] The resulting images are provided in Figure 1A-1F, with Figures 1A-1C being obtained from a sample of white blood cells, and Figures 1D-1F being obtained from a sample of red blood cells infected with malaria.
    [0200] [0200] Specifically, Figure 1A shows in the bright light field image, which shows the borders (the membrane shown by the arrow in Figure 1A) of a white cell and that the DNA staining area (arrow in Figure 1B) and the other stained component area (in this case, the cytoplasm shown in the arrow in Figure 1C) has dimensions that are typical of a white blood cell (both DNA and RNA stains being larger than typical for a Plasmodium parasite).
    [0201] [0201] However, Figures 1D-1F show that the first stained area (DNA, shown by the arrow, Figure 1E) and the second stained area (RNA, white spot in Figure 1F) are much smaller than the cell boundaries (as shown by the arrow in Figure 1D), indicating an anucleated red blood cell infection.
    [0202] [0202] Notably, in an uninfected sample, no stained areas corresponding to those shown in Figure 1E and Figure 1F are obtained.
    [0203] [0203] These results show that, by staining with at least two dyes, one predominantly staining DNA (in this specific case, Hoechst 33342), it is possible to identify Plasmodium infection in a red blood cell. DETECTION CONFIDENCE LEVEL
    [0204] [0204] In this study, a group of 200 blood samples was tested according to one embodiment of the present disclosure. Among these blood samples, 100 were determined positive for malaria using Giemsa stain based on standard microscope methodology and 100 being determined negative. A duplicate of each of the 200 samples was stained with a dye solution to have a final concentration in the tested sample of 2pg / ml Acridine Orange and 15µg / ml Hoechst 33342 and then analyzed using automated machine vision under bright field, UV light and blue light, essentially as described above. The results showed that more than 97% of the samples that were identified as infected under the conventional method were also identified as infected using AO / Hoechst staining. Among the 100 samples that were considered healthy using conventional Giemsa staining, 93 were confirmed using AO / Hoechst staining, but seven samples were identified as infected. These seven were then analyzed again using Giemsa's reagent (repeating both staining and microscopy with a new sample from the same donor or by additional microscopy of the original preparation that gave rise to the negative result). In all seven cases, it was confirmed that they were indeed infected, but apparently in a low parasitemia below 1,000 parasites / μl.
    [0205] [0205] The above shows that not only does the method revealed in this document allow the detection (even by machine vision) of infection with a high level of confidence (above 97%), but also when the parasite count is very low (low parasitemia), even below 1,000 parasites per μl, or below 500 parasites per μl and even 20 parasites / μl.
    权利要求:
    Claims (6)
    [0001]
    METHOD OF DETECTING A PLASMODIUM INFECTION IN A BLOOD SAMPLE, the method characterized by comprising: - the staining of said blood sample with at least Hoechst stain and acridine orange stain; - the acquisition of one or more optical images of the sample; within one or more optical images, identification of at least one colored area that is colored by the Hoechst dye, and at least one other colored area that is colored by the acridine orange dye; and - identification of the blood sample as being infected by plasmodium, identifying, within one or more optical images, at least one among: a colored object comprising at least the first colored area, and the area of the first colored area is in the range of 0.2μm2 to 20μm2; a colored object comprising the first colored area and said at least one other colored area, the area of said at least one other colored area being in the range of 0.8μm2 to 65μm2; and a colored object comprising the first colored area and said at least one other colored area, and the area of the first colored area occupies between 12% and 60% of said at least one other colored area.
    [0002]
    METHOD, according to claim 1, characterized in that the dyes Hoechst and acridine orange are nonspecific to a cell type.
    [0003]
    METHOD according to claim 1, characterized in that staining the blood sample with Hoechst staining comprises staining a blood sample with a Hoechst dye selected from the group consisting of Hoechst 33258, Hoechst 33342, Hoechst 34580, and Hoechst S769121.
    [0004]
    METHOD according to any one of claims 1 to 3, characterized in that said blood sample is a total blood sample and / or a red blood cell sample.
    [0005]
    METHOD, according to any one of claims 1 to 4, characterized by the identification of the blood sample as being infected by plasmodium, comprising identifying the blood sample as being infected by a species of Plasmodium responsible for human malaria, which is selected from the group consisting of P. falciparum, P. vivax, P. ovale and P. malariae.
    [0006]
    SYSTEM FOR DETECTING A PLASMODIUM INFECTION IN A BLOOD SAMPLE, characterized by comprising: - an image capture component configured and operable to acquire at least one optical image of a blood sample; - an image processing unit configured for within at least one optical image, identify at least one first stained area that is stained with a Hoechst stain and at least one other stained area that is stained with acridine orange stain; and identification of the blood sample as infected by plasmodium, identifying, within one or more optical images, at least one among: a colored object comprising at least the first colored area, and the area of the first colored area being in the range of 0.2μm2 to 20μm2; a colored object comprising the first colored area and said at least one other colored area, and the area of said at least one other colored area being in the range of 0.8μm2 to 65μm2; and a colored object comprising the first colored area and said at least one other colored area, and the area of the first colored area occupies between 12% and 60% of said at least one other colored area.
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    同族专利:
    公开号 | 公开日
    CN104169719A|2014-11-26|
    CN106840812A|2017-06-13|
    EP2798350A4|2015-09-02|
    EP2798350A1|2014-11-05|
    CN104169719B|2017-03-08|
    US20150037806A1|2015-02-05|
    EP2798350B1|2021-07-28|
    ZA201405506B|2016-01-27|
    US10640807B2|2020-05-05|
    BR112014016072A8|2017-07-04|
    BR112014016072A2|2017-06-13|
    CN106840812B|2019-12-17|
    WO2013098821A1|2013-07-04|
    US20200181680A1|2020-06-11|
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    法律状态:
    2018-02-14| B25G| Requested change of headquarter approved|Owner name: SIGHT DIAGNOSTICS LTD. (IL) |
    2018-03-27| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|
    2019-08-27| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|
    2020-05-26| B07A| Application suspended after technical examination (opinion) [chapter 7.1 patent gazette]|
    2020-11-17| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|
    2021-01-12| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 27/12/2012, OBSERVADAS AS CONDICOES LEGAIS. |
    优先权:
    申请号 | 申请日 | 专利标题
    ILPCT/IL2011/000973|2011-12-29|
    PCT/IL2011/000973|WO2012090198A2|2010-12-29|2011-12-29|An apparatus and method for automatic detection of pathogens|
    US201261664769P| true| 2012-06-27|2012-06-27|
    US61/664,769|2012-06-27|
    PCT/IL2012/050556|WO2013098821A1|2011-12-29|2012-12-27|Methods and systems for detecting a pathogen in a biological sample|
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