Methods for analyzing fecal matter using fluorochromes that covalently bond to parasitic material

Abstract

Methods of analyzing a fecal sample containing parasitic material and non-parasitic material are disclosed. The method may include combining the fecal sample with a flotation medium in a container. The method may further include at least partially separating the parasitic material and non-parasitic material in the flotation medium. The method may further include extracting at least a portion of the parasitic material from the flotation medium. The method may further include staining or labeling the extracted parasitic material with a fluorochrome comprising a functional group reactive with a functional group of the parasitic material, where the fluorochrome may bind to the parasitic material via a covalent bond. The method may further include placing the labeled parasitic material within an imaging unit for imaging and analysis of the labeled parasitic material.

Description

METHODS FOR ANALYZING FECAL MATTER USING FLUOROCHROMES THAT COVALENTLY BOND TO PARASITIC MATERIALCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Application No. 63 / 658,232, filed June 10, 2024, the entirety of which is incorporated herein by reference.TECHNICAL FIELD

[0002] The present disclosure generally relates to methods of analyzing fecal matter for the presence of parasitic material by separating the parasitic material from non-parasitic material, labeling the parasitic material with a fluorochrome that covalently bonds to the parasitic material, and imaging the labeled parasitic material.BACKGROUND

[0003] Diagnosing internal parasite infection is most commonly achieved by manual microscopic examination of fecal material to ascertain the presence or absence of ova (in the case of helminths) or oocysts (in the case of apicomplexans). This has traditionally been a laborious and inconvenient process, and several methods have been developed over the years. One common theme amongst them is the use of a dense flotation medium (FM), usually a concentrated salt or sugar solution, to separate the parasite ova / oocysts from the bulk of the feces. Since the ova / oocysts are less dense than the medium, they float, while the bulk of the remaining material sinks. Such separation is necessary because the parasite ova / oocysts represent only a tiny fraction of the total feces, and so identifying them visually amongst a field of fecal debris would be problematic, if not impossible.

[0004] In one set of methods (commonly referred to as McMaster methods), floatation occurs passively under gravity in a specialized slide consisting of a sandwich of two slides separated by a gap of 1-2 mm using spacers. A fecal slurry in FM is loaded into the space between the slides and the ova / oocysts are allowed to float to the inner surface of the upper slide. When the slide is examined under a microscope at high power (usually 40-100x), the shallow depth-of-field of the microscope optics has the effect of defocusing the fecal material at the bottom of the chamber when the lens is focused on the ova / oocysts at the top, making them much easier to identify.Methods For Analyzing Fecal Matter Using Fluorochromes That Covalently Bond To Parasitic Material Inventor: Slusarewicz et al Atty Dkt. No. PSGOO 13 WO- 144870-54

[0005] Another set of methods (commonly referred to as Wisconsin of centrifugal float methods) involves centrifugation of fecal slurry in a test tube to speed up the flotation process. Ova / oocysts are collected onto a glass coverslip placed on a meniscus at the surface of the tube. While these methods are more inconvenient and time-consuming than McMaster methods, they tend to have the advantage of greater sensitivity since more feces can be fitted into a test tube than under a McMaster slide.

[0006] More recently advances in digital imaging technologies and deep-learning-based image analysis have led to the development of automated systems for fecal analysis. Automation has the advantage of freeing up analyst time for other tasks since the timeconsuming task of manual examination of specimens is eliminated, ft also reduces or eliminates human error stemming from, for example, inadequate training, fatigue or time-pressure that results in over-hurried examination of the specimen.

[0007] Like manual methods, all current automated methods also rely on either passive or centrifugal floatation to separate the bulk of the feces from the parasite ova / oocysts, and all but one of them also rely on high-magnification image capture of the resulting sample prior to computational analysis. A consequence of this high magnification is a low field-of-view, and so these automated methods must capture large numbers of high magnification, high resolution images in order to analyze the entire area of the sample. This is time consuming with respect to both image collection and analysis, leading to prolonged test times.

[0008] One automated method has bypassed this necessary compromise between magnification and field-of-view by using chemistry to fluorescently label nematode ova with a derivatized recombinant protein (chitin-binding domain; CBD) that binds to the chitin (a linear polysaccharide similar to cellulose) found in the eggshell. The fluorescence imaging modality offers a number of advantages over traditional light microscopy: firstly, labeling chitin enhances the visibility of the parasite ova relative to the bulk of the fecal material; secondly, the high contrast of the images facilitates the capture of a single lower magnification image that nevertheless contain sufficient spatial information to facilitate accurate identification, thereby reducing the computational burden of the system and concomitantly test times; and thirdly, images of samples are captured on a filter mesh (which is also required for staining and washing of the sample), facilitating for the capture and imaging of large amounts of fecal material, thereby increasing test sensitivity.Methods For Analyzing Fecal Matter Using Fluorochromes That Covalently Bond To Parasitic Material Inventor: Slusarewicz et al Atty Dkt. No. PSGOO 13 WO- 144870-54

[0009] While a number of other protein-based ligands have been described that can label some nematode ova in different genera and even differentially bind different species in the same genera, CBD is the only one described that can bind to all helminths. The disadvantages of this approach, however, are: production and conjugation of CBD, like any recombinant protein, is a time-consuming, complex and costly process that increases test costs; CBD does not bind to coccidian oocysts and so cannot be used to detect them; and staining of ova with CBD requires pretreatment with bleach to expose the chitin-containing eggshell. Bleaching not only adds to test time but also poses technical challenges with respect to implementing and dispensing a caustic reagent inside a complex electromechanical device.

[0010] Therefore, there is a need for developing alternative methods for labeling parasite ova / oocysts to facilitate more rapid imaging and analysis while obviating the cost, specificity and chemical disadvantages of CBD.SUMMARY

[0011] The subject matter of the present specification may be understood in terms of the following aspects.

[0012] According to one or more embodiments, methods of analyzing a fecal sample containing parasitic material and non-parasitic material are disclosed. The method may comprise combining the fecal sample with a flotation medium in a container. The method may further comprise at least partially separating the parasitic material and non-parasitic material in the flotation medium. The method may further comprise extracting at least a portion of the parasitic material from the flotation medium. The method may further comprise labeling the extracted parasitic material with a fluorochrome comprising a functional group reactive with a functional group of the parasitic material, where the fluorochrome may bind to the parasitic material via a covalent bond. The method may further comprise placing the labeled parasitic material within an imaging unit for imaging and analysis of the labeled parasitic material.

[0013] Additional features and advantages of the articles described herein will be set forth in the detailed description which follows, and in part will be readily apparent to those skilled in the art from that description or recognized by practicing the embodiments described herein, including the detailed description which follows, the claims, as well as the appended drawings.Methods For Analyzing Fecal Matter Using Fluorochromes That Covalently Bond To Parasitic Material Inventor: Slusarewicz et al Atty Dkt. No. PSGOO 13 WO- 144870-54BRIEF DESCRIPTION OF THE DRAWINGS

[0014] It is to be understood that both the foregoing general description and the following detailed description describe various embodiments and are intended to provide an overview or framework for understanding the nature and character of the claimed subject matter. The accompanying drawings are included to provide a further understanding of the various embodiments, and are incorporated into and constitute a part of this specification. The drawings illustrate the various embodiments described herein, and together with the description serve to explain the principles and operations of the claimed subject matter.

[0015] FIG. 1 A is an exploded view of one or more embodiments of the filter device of the present disclosure;

[0016] FIG. IB is an exploded view of one or more embodiments of the filter device of the present disclosure;

[0017] FIG. 2 shows a plain view of one or more embodiments of the filter device of FIG. 1 prior to insertion into a carrier device containing a dispersion aid;

[0018] FIG. 3 shows an isometric view of a sample collection tool of one or more embodiments of the present disclosure;

[0019] FIG. 4 shows a plain view of the filter device of FIG. 1 as inserted into a carrier device containing a fecal sample pellet according to one or more embodiments of the present disclosure;

[0020] FIG. 5 shows a plain view of a capture filter, a reagent dispensing unit, an imaging unit, and a tablet computer 60 as utilized in one or more embodiments of the methods of the present disclosure;

[0021] FIGS. 6A-6B show entire, single-shot images of the circular 1 cm diameter capture filters from a non-prefiltered sample (FIG. 6A) and a prefiltered sample (FIG. 6B) following fluorescence imaging at lx magnification (i.e. macro) using a camera with a 20-megapixel sensor;

[0022] FIG. 7 shows a series of images of individual stained ova from samples;

[0023] FIG. 8 shows a portion of an image showing stained oocysts identified and circled by the algorithm;Methods For Analyzing Fecal Matter Using Fluorochromes That Covalently Bond To Parasitic Material Inventor: Slusarewicz et al Atty Dkt. No. PSGOO 13 WO- 144870-54

[0024] FIG. 9 shows a graph showing a correlation between the parasitic spike volume and the number of oocysts counted;

[0025] FIGS. 10A-10B show images of stained nematode ova identified and circled;

[0026] FIG. 11 shows an image of stained equine strongylid ova;

[0027] FIG. 12 shows an image of stained canine coccidian oocysts; and

[0028] FIG. 13 shows an image of stained canine coccidian oocysts.

[0029] Reference will now be made in greater detail to various embodiments of the present disclosure, some embodiments of which are illustrated in the accompanying drawings. Whenever possible, the same reference numerals will be used throughout the drawings to refer to the same or similar parts.DETAILED DESCRIPTION

[0030] Described herein are methods for analyzing a fecal sample containing parasitic material and non-parasitic material. In embodiments described herein, fluorochromes comprising a functional group reactive with a functional group of the parasitic material are utilized for staining or labeling the parasite material via a covalent bond to impart fluorescence to the labeled parasitic material that can be imaged. These embodiments are described in detail herein.

[0031] As discussed above, a disadvantage of prior systems is their inability to detect that class of parasitic oocysts as well as ova. Further, there is a need for developing alternative methods for fluorescently labeling parasite ova / oocysts to facilitate more rapid imaging and analysis while obviating the cost, specificity and chemical disadvantages of protein-based stains.

[0032] The present disclosure aims to solve this problem by staining the parasitic material, including oocysts, with a fluorochrome having a functional group that is reactive with a functional group possessed by the parasitic material such that the fluorochrome and parasitic material are joined by a covalent bond. The prior methods utilizing chitin-binding proteins or other protein ligands typically rely upon a combination of hydrogen bonding and hydrophobic interactions.Methods For Analyzing Fecal Matter Using Fluorochromes That Covalently Bond To Parasitic Material Inventor: Slusarewicz et al Atty Dkt. No. PSGOO 13 WO- 144870-54

[0033] In addition, the present disclosure proposes the use of use non-specific, chemically reactive fluorescent moieties to fluoresce all the components of the feces, including any ova or oocysts present. The drawback to this approach is that the non-parasitic components of the feces will also fluoresce (as opposed to when using a stain such as CBD which will only fluoresce ova and insect and fungal fragments, which do not compose the bulk of the sample); these other fecal elements could interfere with detection of ova / oocysts if present in sufficient numbers.

[0034] This possibility could be obviated if less sample was used so that the fecal components were spread less densely across the filter mesh area, or if a larger mesh area was used to the same effect. The disadvantage of the former is that using less sample will reduce the sensitivity of the test, while the problem with the latter is that multiple images of the mesh will need to be collected to capture the entire area, which both complicates the design of the imaging device due to the need for a translational stage and also obviates the advantage of the fluorescence imaging modality (i.e. rapid image capture).

[0035] This disclosure provides methods of processing and staining samples to address these challenges using non-specific, chemically reactive fluorescent dyes to facilitate low- magnification fluorescence imaging for subsequent analysis.

[0036] Various non-limiting embodiments of the present disclosure will now be described to provide an overall understanding of the principles of the structure, function, and use of the apparatuses, systems, methods, and processes disclosed herein. One or more examples of these non-limiting embodiments are illustrated in the accompanying drawings. Those of ordinary skill in the art will understand that systems and methods specifically described herein and illustrated in the accompanying drawings are non-limiting embodiments. The features illustrated or described in connection with one non-limiting embodiment may be combined with the features of other non-limiting embodiments. Such modifications and variations are intended to be included within the scope of the present disclosure.

[0037] Described herein are embodiments of methods for analyzing a fecal sample containing parasitic material and non-parasitic material. The method may comprise at least partially separating the parasitic material and non-parasitic material, extracting the parasitic material, staining the extracted parasitic material with a fluorochrome wherein theMethods For Analyzing Fecal Matter Using Fluorochromes That Covalently Bond To Parasitic Material Inventor: Slusarewicz et al Atty Dkt. No. PSGOO 13 WO- 144870-54 fluorochrome binds to the parasitic material via a covalent bond, and imaging of the labeled parasitic material for analysis of the fluorescent parasitic material.

[0038] In embodiments, the fecal sample may comprise parasitic material and non-parasitic material. The parasitic material may include, but is not limited to, parasitic oocysts, ova, and combinations thereof. As used herein, the term “oocysts” refers to a cyst containing a zygote formed by a parasitic protozoan, such as, for example, apicomplexans. As used herein, the term “ova” refers to eggs of parasitic worms (helminths). The type of parasitic material analyzed include one or more of the following: members of the genus Cooperia, members of the genus Ostertagia, members of the genus Trichostrongylus, members of the genus Haemonchus contortus and members of the genus Bunostomum, members of the genus Strongylus, members of the genus Parascaris, members of the genus Ascaridia, members of the genus Heterakis, members of the genus Dispharynx, members of the genus Syngamus, members of the genus Capillaria, members of the genus Toxocara, members of the genus Ancylostoma, members of the genus Unicinaria, members of the genus Tricuris, members of the genus Oesophagostomum, members of the genus Strongyloides, members of the genus Nematodirus, members of the genus Triodontophorus , members of the genus Cystoisospora, members of the genus Eimeria, members of the genus Giardia, members of the genus Cryptosporidium, members of the genus Monesia, members of the genus Dipylidium, members of the genus Taenia, members of the genus Anoplocephala, members of the genus Fasciola, members of the genus Toxocaris, members of the genus Mesocestoides and members of the genus Physaloptera, among others.

[0039] In embodiments, the method may comprise combining the fecal sample with a flotation medium. The fecal sample and flotation medium may be combined in a container.

[0040] The container may be any suitable container for holding a fluid. In some embodiments, the container may be ajar, cup, beaker, tube, centrifuge tube, vial, or the like.

[0041] In one or more embodiments, the flotation medium may be an aqueous composition comprising water and optionally another component. For example, the flotation medium may comprise an aqueous solution of sodium chloride, sodium nitrate, zinc sulfate, magnesium sulfate, sucrose, glucose, or a combination thereof.Methods For Analyzing Fecal Matter Using Fluorochromes That Covalently Bond To Parasitic Material Inventor: Slusarewicz et al Atty Dkt. No. PSGOO 13 WO- 144870-54

[0042] In one or more embodiments the flotation medium may be selected and / or produced to have a target density. For example, in some embodiments, the parasitic material may have a first density, the non-parasitic material may have a second density, and the flotation medium may have a third density, where the first density, second density, and third density are each different. In some embodiments, the third density of the flotation medium may be greater than the first density of the parasitic material and less than the second density of the non-parasitic material. As discussed above, such a configuration allows the lower density of parasitic material to float in the flotation medium whereas the non-parasitic elements of the bulk feces sink such that the parasitic and non-parasitic materials can be separated when suspended in the flotation medium. The separation can be done passively under gravity or accelerated by centrifugation. In some embodiments, the density of the flotation medium (i.e., third density) may be at least 1.05 g / mL, or at least 1.1 g / mL, or at least 1.2 g / mL. In some embodiments, the density of the flotation medium (i.e., third density) may be no more than 1.3 g / mL, or no more than 1.2 g / mL, or no more than 1.1 g / mL. In some embodiments, the density of the flotation medium (i.e., third density) may be from 1.05 g / mL to 1.3 g / mL, or 1.05 g / mL to 1.2 g / mL, or 1.05 g / mL to 1.1 g / mL, or from 1.1 g / mL to 1.3 g / mL, or 1.1 g / mL to 1.2 g / mL, or 1.2 g / mL to 1.3 g / mL.

[0043] In some embodiments, the method may further comprise at least partially separating the parasitic material and non-parasitic material in the flotation medium. As discussed above, the method of separation is not limited so long as a sufficient amount of non-parasitic material is separated from the parasitic material to allow for staining and imaging of the sample.

[0044] The separating may comprise allowing the parasitic material and non-parasitic material to at least partially separate under gravity due to the differences in density of the materials and the flotation medium. The time for separation under gravity may be any time sufficient to adequately separate the components, such as, for example, 10 minutes or more.

[0045] Optionally, or alternatively, the separation may be more efficiently occur by methods, such as, for example, shaking the container to encourage separation and / or centrifugation of the container to allow the materials to at least partially separate. These optional steps may be done in combination with or in place of gravity -based separation. For centrifugation, centrifuging the sample may be for 0.5 to 10 minutes, or more, and may be from 100 to 10,000 g in a fixed-angle or swing-out centrifuge to float the parasitic material andMethods For Analyzing Fecal Matter Using Fluorochromes That Covalently Bond To Parasitic Material Inventor: Slusarewicz et al Atty Dkt. No. PSGOO 13 WO- 144870-54 sediment the bulk of the non-parasitic material. In one or more embodiments, centrifugation may be from 0.5 to 5 minutes, or from 0.5 to 3 minutes, or from 0.5 to 1.5 minutes, such as 1 minute, and may be from 500 to 5,000 g, or from 1,000 to 3,000 g, or from 1,500 to 2,500 g, such as 2,000 g. The separation of parasitic material in centrifugation is a product of centrifugation time and centripetal force (which is an exponential function of radial velocity). Thus, the same degree sedimentation of fecal material and flotation of parasitic material can be achieved by a vast array of combinations of the two operational parameters. For this reason, centrifugation parameters described in the present disclosure may represent a suitable compromise between these parameters (long spin times will lengthen test times unnecessarily, while faster spin speeds require more costly centrifuges and more time and care to adequately balance sample tubes). As a result, the times / speeds described in the embodiments within should not be considered to be limiting in any way.

[0046] In some embodiments, the method may optionally further comprise filtering non- parasitic material from the flotation medium. The filter is not limited so long as it has pore sizes that allow for retention of at least some of the non-parasitic material and passage of the parasitic material. In some embodiments, the mesh filter may have a pore size of at least 10 microns, or at least 20 microns, or at least 50 microns, or at least 100 microns, or at least 200 microns, or at least 300 microns, or at least 400 microns, or at least 500 microns, or more. In some embodiments, the mesh filter may have a pore size of no more than 1,000 microns, or no more than 800 microns, or no more than 600 microns, or no more than 500 microns, or no more than 400 microns, or no more than 300 microns, or no more than 200 microns, or no more than 100 microns. In some embodiments, the mesh filter may have a pore size of from 10 microns to 1,000 microns, from 10 microns to 800 microns, from 10 microns to 600 microns, from 10 microns to 500 microns, from 10 microns to 400 microns, from 10 microns to 300 microns, from 10 microns to 200 microns, from 10 microns to 100 microns, from 20 microns to 1,000 microns, from 20 microns to 800 microns, from 20 microns to 600 microns, from 20 microns to 500 microns, from 20 microns to 400 microns, from 20 microns to 300 microns, from 20 microns to 200 microns, from 20 microns to 100 microns, from 50 microns to 1,000 microns, from 50 microns to 800 microns, from 50 microns to 600 microns, from 50 microns to 500 microns, from 50 microns to 400 microns, from 50 microns to 300 microns, from 50 microns to 200 microns, from 50 microns to 100 microns, from 100 microns to 1,000 microns, from 100 microns to 800 microns, from 100 microns to 600 microns, from 100 microns to 500Methods For Analyzing Fecal Matter Using Fluorochromes That Covalently Bond To Parasitic Material Inventor: Slusarewicz et al Atty Dkt. No. PSG0013 WO- 144870-54 microns, from 100 microns to 400 microns, from 100 microns to 300 microns, from 100 microns to 200 microns, from 200 microns to 1,000 microns, from 200 microns to 800 microns, from 200 microns to 600 microns, from 200 microns to 500 microns, from 200 microns to 400 microns, from 200 microns to 300 microns, from 300 microns to 1,000 microns, from 300 microns to 800 microns, from 300 microns to 600 microns, from 300 microns to 500 microns, from 300 microns to 400 microns, from 400 microns to 1,000 microns, from 400 microns to 800 microns, from 400 microns to 600 microns, from 400 microns to 500 microns, from 500 microns to 1,000 microns, from 500 microns to 800 microns, from 500 microns to 600 microns. A non-limiting example of a filter device utilizing such filter material is discussed below.

[0047] In embodiments, the method may further comprise extracting at least a portion of the parasitic material from the flotation medium. The method of extraction is not limited so long as a sufficient amount of parasitic material is extracted to be stained and imaged.

[0048] In some embodiments, extracting at least a portion of the parasitic material from the flotation medium may comprise pipetting the parasitic material from a surface of the flotation medium. The pipetted parasitic material may then be transferred to an appropriate container / holder. Although reference is made to “pipetting” the parasitic material, any suitable tool could be used such as, for example, a canula, straw, conduit, etc. Tikewise, the parasitic material may be extracted using a tool to skim the surface, such as a spoon, ladle, sample loop, or the like. The extracted parasitic material optionally may be further filtered to remove extraneous non-parasitic material, if present.

[0049] In one or more embodiments, the method may optionally further comprise pouring the product of the extracting step onto a capture filter. The capture filter may have a pore size sufficiently small enough to capture the parasitic material of interest. In some embodiments, the capture filter may have a pore size of at least 5 microns, or at least 10 microns, or at least 15 microns, or at least 20 microns, or at least 25 microns, or at least 30 microns. In some embodiments, the capture filter may have a pore size of no more than 50 microns, or no more than 40 microns, or no more than 30 microns, or no more than 20 microns, or no more than 10 microns. In some embodiments, the capture filter may have a pore size of from 5 microns to 50 microns, from 5 microns to 40 microns, from 5 microns to 30 microns, from 5 microns to 20 microns, from 5 microns to 10 microns, from 10 microns to 50 microns, from 10 microns to 40 microns, from 10 microns to 30 microns, from 10 microns to 20 microns, from 15 micronsMethods For Analyzing Fecal Matter Using Fluorochromes That Covalently Bond To Parasitic Material Inventor: Slusarewicz et al Atty Dkt. No. PSGOO 13 WO- 144870-54 to 50 microns, from 15 microns to 40 microns, from 15 microns to 30 microns, from 15 microns to 20 microns, from 20 microns to 50 microns, from 20 microns to 40 microns, from 20 microns to 30 microns, from 25 microns to 50 microns, from 25 microns to 40 microns, from 25 microns to 30 microns, from 30 microns to 50 microns, from 30 microns to 40 microns.

[0050] In embodiments, the method may further comprise staining the extracted parasitic material with a fluorochrome comprising a functional group reactive with a functional group of the parasitic material, wherein the fluorochrome binds to the parasitic material via a covalent bond to turn the parasitic material into fluorescent parasitic material. For the purposes of this disclosure, the term “fluorochrome” refers to any kind of molecular or macromolecular structure that is fluorescent (including aggregates, colloids and nanoparticles), while the term “fluorophore” refers to solely to fluorescent small molecules. The fluorochrome is not limited so long as it has a functional group capable of reacting with a functional group of the parasitic material to bind to the parasitic material via a covalent bond. For example, the functional group of the fluorochrome may comprise an epoxide, a fluorophenyl ester, an isocyanate, an isothiocyanate, a N-hydroxysuccinimide, a carbodiimide, an anhydride, a sulfonyl chloride, an aldehyde, an oxirane, an acyl azide, an aryl halide, an imidoester, a glyoxal, or any combination thereof. Likewise, the functional group of the parasitic material may be any functional group found in biological structures, such an amine, such as a primary amine, a thiol, a carboxylic acid, an alcohol, an aldehyde, or combinations thereof.

[0051] In some embodiments, the fluorochrome may comprise a chemical fluorophore, a nanoparticle, a colloidal particle, a quantum dot, or a combination thereof. In one of more embodiments, the chemical fluorophore may be comprise, but is not limited to, fluorescein, fluorescein isothiocyanate (FITC), cyanine dyes (Cy3, Cy3.5, Cy5, Cy5.5, Cy7), Bodipy and / or Alexa Fluor dyes, dansyl, dansyl chloride (DNS-C1), 5-(iodoacetamida)fluorescein (5-IAF, 6- acryloyl]-2-dimethylaminonaphthalene (acrylodan), 7-nitrobenzo-2-oxa-l ,3,-diazol-4-yl chloride (NBD-C1), ethidium bromide, Lucifer Yellow, rhodamine dyes (5-carboxyrhodamine 6G hydrochloride, lissamine rhodamine B; tetramethylrhodamine 5-(and 6-)isothiocyanate (TRITC)), Texas Red™, sulfonyl chloride, naphthalamine sulfonic acids including but not limited to l-anilinonaphthalene-8-sulfonic acid (ANS) and 6-(p-toluidinyl)naphthalene-2- sulfonic acid (TNS), anthroyl fatty acid, DPH, parinaric acid, TMA-DPH, fluorenyl fatty acid, fuorescein-phosphatidylethanolamine, Texas red-phosphatidylethanolamine, pyrenyl-Methods For Analyzing Fecal Matter Using Fluorochromes That Covalently Bond To Parasitic Material Inventor: Slusarewicz et al Atty Dkt. No. PSGOO 13 WO- 144870-54 phophatidyl-choline, Fluorenyl-phosphatidylcholine, Merocyanine 540, naphtyl styryl, 3,3'dipropylthiadicarbocyanine (diS-C3-(5)), 4-(p-dipentyl aminostyryl)- 1 -methylpyridinium (di-5-ASP), Cy-3 Iodo Acetamide, Cy-5-N-Hydroxysuccinimide, Cy-7-Isothiocyanate, IR- 125, Thiazole Orange, azure B, Nile Blue, Al Phthalocyanine, oxaxine l,4',6-diamidino-2- phenylindole. (DAPI), Hoechst 33342, TOTO, acridine orange, ethidium homodimer, N(ethoxycarbonylmethyl)-6-methoxyquinolinium (MQAB), Fura-2, calcium green, carboxy SNARF-6, BAPTA, coumarin, phyto fluors, coronene, and metal-ligand complexes.

[0052] In one or more embodiments, the fluorochrome may be unstable in aqueous solvent due to their reactive functional groups. Accordingly, in one or more embodiments, the fluorochrome may be stored and transported as lyophilized aliquots of the fluorochromes to be reconstituted with an appropriate solvent at the time of use. Alternatively, in one or more embodiments, a solution of the reactive fluorochrome in a suitable solvent may be used to yield a satisfactory shelf-life. Any suitable solvent may be used for this purpose. In some embodiments, the solvent may be a water-miscible, polar aprotic solvent. Non-limiting examples of suitable solvents include, but are not limited to, acetone, acetonitrile (ACN), dichloromethane, dimethylacetamide, dimethyl formamide (DMF), N-methylpyrrolidone, dimethylimidazolidone, dimethylpropyleneurea, dimethyl sulfoxide (DMSO), ethyl acetate, hexamethylphosphoramide, propylene carbonate, pyridine, sulfolane or tetrahydrofuran, or a combination thereof. Such solvents may reduce hydrolysis and thus increase shelf-life. The fluorochrome in solvent optionally may be mixed with an aqueous medium prior to using it to stain the parasitic material, or, alternatively, the fluorochrome in solvent may mix with an aqueous medium upon addition to the parasitic material for the purposes of staining the parasitic material. In some embodiments, the concentration of the fluorochrome when combined with the parasitic material for staining may be at least 25 pg / mL, or higher, and may go up to the solubility limit of the fluorochrome, although very high levels may require more washing to remove excess fluorochrome.

[0053] In one or more embodiments, staining or labeling the extracted parasitic material with a fluorochrome may be done with the parasitic material on the capture filter. In some embodiments, the fluorochrome dissolved in solvent is added to the parasitic material on the capture filter followed sequentially by addition of an aqueous medium. In some embodiments,Methods For Analyzing Fecal Matter Using Fluorochromes That Covalently Bond To Parasitic Material Inventor: Slusarewicz et al Atty Dkt. No. PSGOO 13 WO- 144870-54 the aqueous medium is added to the parasitic material on the capture filter followed sequentially by addition of the fluorochrome dissolved in solvent.

[0054] In one of more embodiments, the method may optionally further comprise treating the parasitic material with a buffer to change the pH prior to or concurrent with staining. The buffer may be an aqueous buffer. In some embodiments, the buffer may be neutral. In other embodiments, the buffer may be alkaline. The buffer used may depend upon the functional groups of the target parasitic material that the fluorochrome is to react with. For example, the buffer may be alkaline when the reactive groups of the parasitic material include amines, and the buffer may be alkaline or neutral when the reactive groups of the parasitic material include thiols. The buffer may be used to treat the parasitic material before the parasitic material is stained or may be added contemporaneously thereto in order to improve reaction conditions. For example, the buffer may be premixed with the fluorochrome and solvent prior to adding to the parasitic material, such as in the capture filter. Non-limiting examples of suitable buffer solutions include, but are not limited to, sodium bicarbonate, sodium carbonate, potassium bicarbonate, potassium carbonate, potassium phosphate, or sodium phosphate. The buffers described can be used to maintain the optimal pH during the course of the reaction. The buffers described should not be considered limiting, since any number of different buffers could be used for any given pH that could be selected by one of ordinary skill in the art. In addition, any number of buffer concentrations could be selected for this purpose, and so the concentrations in the embodiments disclosed here should not be considered limiting. Finally, in cases of neutral pH, a buffer may be omitted.

[0055] In one or more embodiments, the method may omit any bleaching of the parasitic material prior to staining such that the parasitic material is not bleached prior to staining. As discussed above, prior techniques required bleach to expose the chitin-containing eggshell of the parasitic material. However, bleaching adds to test time and also poses technical challenges with respect to implementing and dispensing a caustic reagent inside a complex electromechanical device.

[0056] In one or more embodiments, the method may further comprise washing excess fluorochrome from the parasitic material after staining. The washing may be with water.Methods For Analyzing Fecal Matter Using Fluorochromes That Covalently Bond To Parasitic Material Inventor: Slusarewicz et al Atty Dkt. No. PSGOO 13 WO- 144870-54

[0057] In one or more embodiments, the method may further comprise placing the fluorochrome-labeled parasitic material within an imaging unit for imaging and analysis of the fluorescent parasitic material. The imaging unit may be any suitable imaging unit for fluorescence imaging, such as that described in the Examples section herein. Likewise, the analysis of the image may be any suitable technique, such as those described in the Examples section herein.

[0058] In one of more embodiments, a filter device may be used to filter and separate the parasitic material and non-parasitic material. The filter device is not limited and various filter devices are known to those of skill in the art. For example, in one or more embodiments, the filter device may be according to the filter device described below and referred to in FIGS. 1- 4. However, the filter device is not so limited and may include other filter devices, including, for example, pour-through filters, bag filters, cartridge filters, membrane filters, and the like, in any suitable configuration so long as the configuration allows for at least partial separation of the parasitic material and non-parasitic material in a way that allows for the extraction of the parasitic material.

[0059] In one of more embodiments, the exemplary filter device described below and exemplified by the attached drawings may be used in the method of the present disclosure. Specifically, a filter device 10 as shown in FIGS. 1A and IB may be utilized. The filter device 10 includes a separator tube 12, an O-ring or other type of gasket 14, and a mesh filter 16. The mesh filter 16 may be attached to the separator tube 12. In one or more embodiments, the means of attachment include the use of an adhesive, by heat staking or by sonic welding. In one or more embodiments, the filter device 10 also contains one or more notches 18 on the separator tube 12 at the opposite end of the tube 12 to the mesh filter 16. The one or more notches 18 allow air to escape when the filter device 10 is pressed into a sample tube. The length of the separator tube 12 needs to be long enough so as to reach below the level of the least-floated target parasitic material (e.g., ovum / oocyst), but ideally short enough so as not to disturb the sedimented fecal material when the device 10 is pressed into a sample tube. In one or more embodiments, the length of the separator tube 12 may be between about 2 and about 4 inches. In one embodiment, the length of the separator tube 12 may be 3 inches. In another embodiment, the device 10 contains a flange (not shown) instead of the O-ring 14. IfMethods For Analyzing Fecal Matter Using Fluorochromes That Covalently Bond To Parasitic Material Inventor: Slusarewicz et al Atty Dkt. No. PSGOO 13 WO- 144870-54 present, the flange serves the same purpose as the O-ring 14, which is to prevent loss of sample between the sample tube and the tube 12 of device 10.

[0060] FIG. 1A shows an embodiment of the filter device 10 wherein the O-ring 14 is placed within a groove G adjacent the end of the filter device 10. FIG. IB shows an embodiment of the filter device 10 wherein instead of a groove G adjacent an end of the filter device, the end of the filter device is stepped such that a step S is created. In the embodiment shown in FIG. IB, the O-ring 14 should be slid over the step S prior to the mesh filter 16 being attached to the filter device 10.

[0061] Other components needed for the method of the present disclosure are shown in FIG. 2 and include a sample centrifuge tube 20 and a dispersion aid 22, such as the ball bearing shown. The dispersion aid 22 can either be disposable or reusable. In one or more embodiments, the dispersion aid 22 incorporates fins or blades to aid in sample dispersion. In one or more embodiments, the centrifuge tube 20 is a 15 mL tube. In one or more embodiments, the centrifuge tube 20 can be supplied to the end user preloaded with between 5 and 14 mL of a flotation medium, such as, but not limited to sodium nitrate, sodium chloride, sugar (such as sucrose) a sugar salt mix, magnesium sulfate or zinc sulfate. In one embodiment, the tube 20 is preloaded with 10 mL of a flotation medium. If not preloaded, between 5 and 14 mL of a flotation medium should be added to the centrifuge tube 20 prior to any fecal sample being added to the tube 20. In another embodiment, the tube is preloaded with the dispersion aid either with or without the floatation medium.

[0062] To analyze a fecal sample containing parasitic material and non-parasitic material according to one or more embodiments of the present disclosure, a fecal sample will need to be placed in the centrifuge tube 20. In one or more methods of the present disclosure, the fecal sample is prepared by utilizing a sample collection tool 24 as shown in FIG. 3. The sample collection tool 24 gathers a small fecal sample from the collected fecal sample, and then the small fecal sample is deposited into the tube 20. In one or more embodiments, the sample collection tool 24 contains a plunger 26 that can be utilized to assist in depositing the small fecal sample into the tube 20. To fill the sample collection tool 24, the plunger 26 should be pulled back, which will meter the collection. Then, once the tool 24 is filled with a fecal sample, the plunger 26 can be depressed for easy ejection of the sample from the tool 24 into the tube 20. Once the sample has been added to the tube 20, the dispersion aid 22 can then beMethods For Analyzing Fecal Matter Using Fluorochromes That Covalently Bond To Parasitic Material Inventor: Slusarewicz et al Atty Dkt. No. PSGOO 13 WO- 144870-54 added to the tube 20, and the tube 20 is then capped with any conventional tube cap. It is also contemplated that the dispersion aid 22 can be added to the tube 20 prior to introduction of the fecal sample.

[0063] Once capped, the centrifuge tube 20 may be vigorously shaken to begin the process of mixing the fecal sample with the flotation medium. This shaking may assist in breaking apart the fecal sample. Once shaken, in some embodiments of the present disclosure, the centrifuge tube 20 may then then be added to a centrifuge. The tube 20 may then be centrifuged with a counter balance for a time period of about between about 30 seconds and 5 minutes at a speed of between about 500 g and 3,000 g. In one embodiment, the tube 20 is centrifuged for 1 minute at 2,000 g. In other embodiments, centrifugation is not needed, and after the period of shaking, the contents of the tube 20 are allowed to just settle on their own for a time period of between about 5 minutes and 60 minutes. However, without centrifugation, the results of the method will take longer to collect due to the prolonged standing time.

[0064] Either after a standing period or the end of the centrifugation, the tube 20 may then be uncapped. The tube 20 will then have a pellet of matter P present at the bottom of tube 20. Next, the filter device 10 is pressed into the tube 20. The filter device 10 should be inserted with the mesh filter 16 entering the tube 20 first. The filter device 10 should be placed at a depth of between about 0.25 and about 6 inches below the surface of the liquid within the tube 20. In one or more embodiments, when using an imaging system as discussed above, once the filter device 10 is in place, the sample captured within the tube 12 is then poured onto a capture filter 30, such as a chamber filter as shown in FIG. 5. In one or more embodiments, the chamber 30 is then placed within a reagent dispensing unit 40 and then an imaging unit 50 as shown in FIG. 5. Although the fluorochrome dispensing unit 40 and the imaging unit 50 are shown in two different housings, it is also contemplated that both units can be housed within one singular unit. The fluorochrome dispensing unit 40 will dispense a fluorochrome that allow for ova and oocysts to fluoresce, which will then allow for the imaging unit 50 to take better images for the ova and oocysts. The images taken by the imaging unit 50 can then be viewed on any computing device, such as the computing device 60 shown in FIG. 5. The computing device 60 will also run a counting algorithm in one or more embodiments.

[0065] In another embodiment, the filter device 10 can be placed into the tube 20 prior to shaking or after shaking but before the optional centrifugation. In this embodiment, large fecalMethods For Analyzing Fecal Matter Using Fluorochromes That Covalently Bond To Parasitic Material Inventor: Slusarewicz et al Atty Dkt. No. PSGOO 13 WO- 144870-54 particles are forced downwards while the ova and oocysts pass through the mesh filter 16 into the device 10 during shaking and the optional centrifugation. During shaking and the optional centrifugation, any fecal material pressed against the mesh filter 16 will sink downwards, thereby unclogging the mesh filter 16 and allowing the ova and oocysts below the mesh filter 16 to flow upwards and through the mesh filter.

[0066] When the device 10 is pressed into the centrifuge tube 20, the mesh filter 16 separates the bulk fecal matter at the bottom of tube 20 from the liquid containing the floating ova and oocysts at the top of the tube 20. The ova and oocysts float due to the density of the ova and oocysts as compared to the density of the floatation medium within the tube 20. This separation by device 10 will prevent clumps of fecal matter that may dislodge from the pellet P from entering into the capture filter 30 when the sample is poured therein. The pores of the mesh filter 16 therefore need to be sufficiently large enough to allow the ova and oocysts to pass through, but small enough to prevent the liquid below it from flowing through by virtue of the surface tension created by insertion of the device 10 into the tube 20. In other words, the mesh filter 16 does not simply prevent larger fecal particle from passing through, but rather the mesh filter 16 prevents all of the liquid and solid material located beneath the mesh filter 16 from passing through. In one or more embodiments, the mesh filter should have a pore size of between about 80 microns and about 1,000 microns. In the case of samples that contain smaller parasite products such as coccidian oocysts, the pore size can be as low as 20-40 microns.

[0067] The purpose of the filter device is to separate the bulk feces of the pellet of matter P at the bottom of the tube 20 from the liquid (containing floating parasitic material) above it. This will prevent clumps of feces that may dislodge from the pellet P from entering the capture filter 30. The pores of the mesh filter 16 of the filter device 10 therefore need to be sufficiently large to allow ova and oocysts to pass though, but small enough to prevent the liquid below it from flowing through by virtue of the surface tension of the mesh filter 16. That is to say, the purpose of the mesh filter 16 is not simply to prevent larger particles from passing through, but rather to prevent all the liquid and solid beneath it from doing so. In one or more embodiments, the mesh filter 16 may have pore sizes between 20 and 1000 microns. The device 10 will also assist in stopping the dispersion aid 22 from falling onto the capture filter 30. In one or more embodiments, if the animal that produced the sample is a ruminant, such as a cow, a sheep, orMethods For Analyzing Fecal Matter Using Fluorochromes That Covalently Bond To Parasitic Material Inventor: Slusarewicz et al Atty Dkt. No. PSGOO 13 WO- 144870-54 a goat, the pore size of the capture filter 30 may be between 20 microns and 40 microns, and in an embodiment the pore size of the capture filter may be 25 microns. In one embodiment, if the animal that produced the sample is a cat or a dog, the pore size of the capture filter 30 may be between 15 microns and 25 microns, and in one embodiment the pore size of the capture filter may be 20 microns. In one or more embodiments, if the animal that produced the sample is an equine, the pore size of the mesh filter 16 may be between 30 microns and 50 microns, and in one embodiment the pore size of the capture filter may be 37 microns.

[0068] The terms “free” and “substantially free,” when used to describe the concentration and / or absence of a particular constituent component means that the constituent component is not intentionally added.

[0069] Ranges can be expressed herein as from “less than or equal to” one particular value, and / or to “less than or equal to” another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “less than or equal to,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. Any ranges used herein include all ranges and subranges and any values there between unless explicitly stated otherwise.

[0070] Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.

[0071] Directional terms as used herein - for example up, down, right, left, front, back, top, bottom - are made only with reference to the figures as drawn and are not intended to imply absolute orientation.

[0072] Unless otherwise expressly stated, it is in no way intended that any method set forth herein be construed as requiring that its steps be performed in a specific order, nor that withMethods For Analyzing Fecal Matter Using Fluorochromes That Covalently Bond To Parasitic Material Inventor: Slusarewicz et al Atty Dkt. No. PSGOO 13 WO- 144870-54 any apparatus specific orientations be required. Accordingly, where a method claim does not actually recite an order to be followed by its steps, or that any apparatus claim does not actually recite an order or orientation to individual components, or it is not otherwise specifically stated in the claims or description that the steps are to be limited to a specific order, or that a specific order or orientation to components of an apparatus is not recited, it is in no way intended that an order or orientation be inferred, in any respect. This holds for any possible non-express basis for interpretation, including: matters of logic with respect to arrangement of steps, operational flow, order of components, or orientation of components; plain meaning derived from grammatical organization or punctuation, and; the number or type of embodiments described in the specification.

[0073] As used herein, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a” component includes aspects having two or more such components, unless the context clearly indicates otherwise.

[0074] Reference throughout the specification to “various embodiments,” “some embodiments,” “one embodiment,” “some example embodiments,” “one example embodiment,” or “an embodiment” means that a particular feature, structure, or characteristic described in connection with any embodiment is included in at least one embodiment. Thus, appearances of the phrases “in various embodiments,” “in some embodiments,” “in one embodiment,” “some example embodiments,” “one example embodiment,” or “in an embodiment” in places throughout the specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner in one or more embodiments.

[0075] A “computer,” “computer system,” “host,” “server,” or “processor” can be, for example and without limitation, a processor, microcomputer, minicomputer, server, mainframe, laptop, personal data assistant (PDA), wireless e-mail device, cellular phone, pager, processor, fax machine, scanner, or any other programmable device configured to transmit and / or receive data over a network. Computer systems and computer-based devices disclosed herein can include memory for storing certain software modules used in obtaining, processing, and communicating information. It can be appreciated that such memory can be internal or external with respect to operation of the disclosed embodiments. The memory can also include any means for storing software, including a hard disk, an optical disk, floppy disk, ROM (readMethods For Analyzing Fecal Matter Using Fluorochromes That Covalently Bond To Parasitic Material Inventor: Slusarewicz et al Atty Dkt. No. PSG0013 WO- 144870-54 only memory), RAM (random access memory), PROM (programmable ROM), EEPROM (electrically erasable PROM) and / or other computer-readable media. Non-transitory computer- readable media, as used herein, comprises all computer-readable media except for a transitory, propagating signals.

[0076] In various embodiments disclosed herein, a single component can be replaced by multiple components and multiple components can be replaced by a single component to perform a given function or functions. Except where such substitution would not be operative, such substitution is within the intended scope of the embodiments.

[0077] The examples discussed herein are examples only and are provided to assist in the explanation of the apparatuses, devices, systems and methods described herein. None of the features or components shown in the drawings or discussed below should be taken as mandatory for any specific implementation of any of these the apparatuses, devices, systems or methods unless specifically designated as mandatory. For ease of reading and clarity, certain components, modules, or methods may be described solely in connection with a specific figure. Any failure to specifically describe a combination or sub-combination of components should not be understood as an indication that any combination or sub-combination is not possible. Also, for any methods described, regardless of whether the method is described in conjunction with a flow diagram, it should be understood that unless otherwise specified or required by context, any explicit or implicit ordering of steps performed in the execution of a method does not imply that those steps must be performed in the order presented but instead may be performed in a different order or in parallel.

[0078] Having shown and described various versions in the present disclosure, further adaptations of the methods and systems described herein may be accomplished by appropriate modifications by one of ordinary skill in the art without departing from the scope of the present disclosure. Several of such potential modifications have been mentioned, and others will be apparent to those skilled in the art. For instance, the examples, versions, geometries, materials, dimensions, ratios, steps, and the like discussed above are illustrative and are not required. Accordingly, the scope of the present disclosure should be considered in terms of the following claims and is understood not to be limited to the details of structure and operation shown and described in the specification and drawings.Methods For Analyzing Fecal Matter Using Fluorochromes That Covalently Bond To Parasitic Material Inventor: Slusarewicz et al Atty Dkt. No. PSGOO 13 WO- 144870-54EXAMPLES

[0079] The various embodiments of systems and processes of the present disclosure will be further clarified by the following examples. The examples are illustrative in nature, and should not be understood to limit the subject matter of the present disclosure.

[0080] The subject of this disclosure is the process for staining a fecal sample in preparation for fluorescence imaging and analysis, and not the imaging and analysis per se. However, algorithm used to evaluate these, as well as the other images in the embodiments described in this disclosure is a YOTO (you only look once)-type algorithm trained on a curated, manually annotated dataset of thousands of images of different ova / oocyst types. This and other similar algorithms are publicly available and can readily be utilized by anyone of ordinary skill in the art to analyze images similar to those presented here.

[0081] Example 1 : Two 1 g portions of the same negative fecal sample were each dispersed in 10 mE of a 1.1 g / L flotation medium (FM) including sodium nitrate and spiked with the same volume (20 pl) of purified canine coccidian oocysts. The suspensions were placed in separate tubes and centrifuged at 2,000 g for 1 minute. One supernatant was then passed through a mesh filter with a pore size of 43 microns and poured onto a capture mesh filter with a pore size of 20 microns. The other supernatant was poured onto a separate 43-micron capture mesh filter without prefiltering. Once the liquid was pulled through under vacuum, two drops of 100 mM sodium bicarbonate buffer at pH 10 (SBB) were dispensed onto the capture mesh followed by two drops of 1.275 mg / mE fluorescein isothiocyanate (FITC) in acetonitrile and then another two drops of SBB. The mixture was left to stand for 3 minutes at room temperature, at which point it was removed by vacuum and washed 9 times with 150 microliters of SBB. Fig. 6 shows both entire, single-shot images of the circular 1 cm diameter capture filters from both samples following fluorescence imaging at lx magnification (i.e. macro) using a camera with a 20-megapixel sensor. The insets show expanded views of selected areas of the full-size images. The sample that was not prefiltered (Fig. 6A) exhibits vastly more fecal debris than the corresponding image of the filtered sample (Fig. 6B). Furthermore, the debris in the unfiltered sample either completely obscures or makes oocysts more difficult to observe (see insets). The algorithm identified only 3 oocysts in the image from the unfiltered sample and 240 oocysts in the image from the unfiltered sample, even though both samples were spiked with the same volume of the purified oocyst suspension.Methods For Analyzing Fecal Matter Using Fluorochromes That Covalently Bond To Parasitic Material Inventor: Slusarewicz et al Atty Dkt. No. PSGOO 13 WO- 144870-54

[0082] Example 2: Fecal samples were similarly prepared and spiked as in Example 1 but instead were treated with varying concentrations of FITC using a series of 1 : 1 dilutions with acetonitrile from an initial stock of 1.275 mg / mL FITC in acetonitrile. Fig. 7 shows a series of images of individual ova from these samples. It should be noted that the concentrations shown are those of the acetonitrile stocks; since the samples are diluted 3-fold by the addition of SBB, the actual concentrations of FITC upon addition of the ova was one third of the concentrations indicated in Fig. 2.

[0083] The intensity of the images of the parasitic material in the samples decreased with decreasing FITC concentration. While they were still discernable by the naked eye at a 1 :8 dilution (160 pg / mL as an acetonitrile stock or 53 pg / mL in the reaction), below this level they became too dim to observe without first artificially enhancing the brightness using Adobe Photoshop software. At a 1 :64 dilution, oocysts could not be observed at all. The algorithm was able to detect large numbers of ova at all the dilutions until the 1 :16 dilution, at which point it detected no ova at all. Thus oocysts can be successfully fluoresced by concentration of greater than 25 pg / mE. However, since higher concentrations had no adverse effects on staining patterns, high concentrations can be use to provide a buffer against degradation of the FITC during storage.

[0084] Example 3: A negative canine fecal sample spiked with 20 pl of purified canine coccidia oocysts was processed as in Examples 1 and 2 using a stock of 1.275 mg / mL FITC in acetonitrile. The image thus generated was analyzed by an algorithm trained with similarly produced images. The algorithm successfully detected the oocysts in the image. A portion of the image showing oocysts identified and circled by the algorithm is shown in Fig. 8.

[0085] Example 4 : A number of 1 g portions of negative canine fecal sample were spiked with 5, 10, 15, 20 or 25 pL of purified canine coccidia. The samples were treated as described in Example 3. The resulting images were processed with the same algorithm and the correlation between the spike volume and the number of oocysts counted is shown in Fig. 9. The strong correlation indicated that this approach produced not only qualitative but also quantitative results.

[0086] Example 5 : Two canine fecal samples that were positive for both Ancylostoma and Trichiiris or positive for Toxocara nematode parasites were examined. One g of each sampleMethods For Analyzing Fecal Matter Using Fluorochromes That Covalently Bond To Parasitic Material Inventor: Slusarewicz et al Atty Dkt. No. PSGOO 13 WO- 144870-54 was individually suspended in 10 mL of a 1.18 g / L FM including sodium nitrate. The suspensions were placed in separate tubes and centrifuged at 2,000 g for 1 minute. Each supernatant was then passed through a mesh filter with a pore size of 130 microns and poured onto a capture mesh filter with a pore size of 20 microns. Once the liquid was pulled through under vacuum, two drops of 100 rnM sodium bicarbonate buffer at pH 10 (SBB) were dispensed onto the capture mesh followed by two drops of 1.275 mg / mL fluorescein isothiocyanate (FITC) in acetonitrile and then another two drops of SBB. The mixture was left to stand for 3 minutes at room temperature, at which point it was removed by vacuum and washed 9 times with 150 microliters of SBB prior to imaging. The FITC labeled all three types of nematode ova (Fig. 10A and 10B). While the brightness intensities of these ova were substantially lower than for ova labeled with a more specific dye (i.e., CBD) they could still be easily distinguished from the background material despite the specificity of the FITC staining being substantially lower than that of CBD. Furthermore, an algorithm trained on nematode ova stained with CBD was still able to both identify and differentiate between the different types of ova.

[0087] Example 6: One g of an equine fecal sample containing strongylid ova was suspended in 10 mL of a 1.1 g / L FM including sodium nitrate. The suspension was placed in a tube and centrifuged at 2,000 g for 1 minute. The supernatant was then passed through a mesh filter with a pore size of 130 microns and poured onto a capture mesh filter with a pore size of 20 microns. Once the liquid was pulled through under vacuum, two drops of 100 mM sodium bicarbonate buffer at pH 10 (SBB) were dispensed onto the capture mesh filter followed by two drops of 1.275 mg / mL fluorescein isothiocyanate (FITC) in acetonitrile and then another two drops of SBB. The mixture was left to stand for 3 minutes at room temperature, at which point it was removed by vacuum and washed 9 times with 150 microliters of SBB prior to imaging. Similarly to the canine nematode-positive samples, FITC labeled equine strongylid ova (Fig. 11). Also similarly, an algorithm trained on equine nematode ova stained with CBD was still able to both identify these FITC-stained equine strongylid ova.

[0088] Example 7 : One g of a negative canine fecal sample spiked with 20 pL of purified canine coccidia was suspended in 10 mL of a 1.1 g / L FM including sodium nitrate. The suspension was placed in a tube and centrifuged at 2,000 g for 1 minute. The supernatant was then passed through a mesh filter with a pore size of 43 microns and poured onto a captureMethods For Analyzing Fecal Matter Using Fluorochromes That Covalently Bond To Parasitic Material Inventor: Slusarewicz et al Atty Dkt. No. PSGOO 13 WO- 144870-54 mesh filter with a pore size of 20 microns. Once the liquid was pulled through under vacuum, two drops of 100 mM sodium bicarbonate buffer at pH 10 (SBB) were dispensed onto the capture mesh filter followed by two drops of 1.275 mg / mL of 5 / 6-carboxy fluorescein succinimidyl ester (NHS-FL) in acetonitrile and then another two drops of SBB. The mixture was left to stand for 3 minutes at room temperature, at which point it was removed by vacuum and washed 9 times with 150 microliters of SBB prior to imaging. Like FITC, NHS-FL also reacts with deprotonated primary amines, and also strongly stained canine coccidian oocysts (Fig. 12). The same algorithm trained on FITC-stained oocysts also recognized these oocysts.

[0089] Example 8: One g of a negative canine fecal sample spiked with 20 pL of purified canine coccidia was suspended in 10 mL of a 1.1 g / L FM including sodium nitrate. The suspension was placed in a tube and centrifuged at 2,000 g for 1 minute. The supernatant was then passed through a mesh filter with a pore size of 43 microns and poured onto a capture mesh filter with a pore size of 20 microns. Once the liquid was pulled through under vacuum, two drops of phosphate -buffered saline (PBS) were dispensed onto the capture mesh followed by two drops of 1.275 mg / mL of 5 / 6-carboxy fluorescein succinimidyl ester (NHS-FL) in acetonitrile and then another two drops of PBS. The mixture was left to stand for 3 minutes at room temperature, at which point it was removed by vacuum and washed 9 times with 150 microliters of PBS prior to imaging. Unlike FITC and NHS-FL, fluorescein-maleimide can react with both deprotonated primary amines and thiols. However, at neutral pH, such as that of PBS, amines become protonated and fluorescein-maleimide reacts only with thiols. Such were the conditions in this embodiment, which demonstrated that thiol-based staining does stain oocysts, albeit more faintly, though strongly enough for them to still be recognized by the algorithm trained on FITC-stained oocysts (Fig. 13).

[0090] ft is noted that any two quantitative values assigned to a property may constitute a range of that property, and all combinations of ranges formed from all stated quantitative values of a given property are contemplated in this disclosure.

[0091] ft is noted that one or more of the following claims utilize the term "where" as a transitional phrase. For the purposes of defining the present technology, it is noted that this term is introduced in the claims as an open-ended transitional phrase that is used to introduce a recitation of a series of characteristics of the structure and should be interpreted in like manner as the more commonly used open-ended preamble term "comprising."Methods For Analyzing Fecal Matter Using Fluorochromes That Covalently Bond To Parasitic Material Inventor: Slusarewicz et al Atty Dkt. No. PSGOO 13 WO- 144870-54

[0092] Having described the subject matter of the present disclosure in detail and by reference to specific aspects, it is noted that the various details of such aspects should not be taken to imply that these details are essential components of the aspects. Rather, the claims appended hereto should be taken as the sole representation of the breadth of the present disclosure and the corresponding scope of the various aspects described in this disclosure. Further, it will be apparent that modifications and variations are possible without departing from the scope of the appended claims.

Claims

Methods For Analyzing Fecal Matter Using Fluorochromes That Covalently Bond To Parasitic Material Inventor: Slusarewicz et al Atty Dkt. No. PSGOO 13 WO- 144870-54CLAIMSWhat is claimed is:

1. A method of analyzing a fecal sample containing parasitic material and non-parasitic material, the method comprising: combining the fecal sample with a flotation medium in a container; at least partially separating the parasitic material and the non-parasitic material in the flotation medium; extracting at least a portion of the parasitic material from the flotation medium; labeling the extracted parasitic material with a fluorochrome comprising a functional group reactive with a functional group of the parasitic material, wherein the fluorochrome binds to the parasitic material via a covalent bond; and placing the labeled parasitic material within an imaging unit for imaging and analysis of the labeled parasitic material.

2. The method of claim 1, wherein at least partially separating the parasitic material and non-parasitic material in the flotation medium comprising allowing the parasitic material and non-parasitic material to at least partially separate under gravity or by centrifugation.

3. The method of claim 1, wherein the parasitic material has a first density, the non- parasitic material has a second density, the flotation medium has a third density, and the third density is greater than the first density and less than the second density.

4. The method of claim 1, wherein the flotation medium has a density of from 1.05 g / mT and 1.3 g / mT.

5. The method of claim 1, wherein the functional group of the fluorochrome comprises an epoxide, a fluorophenyl ester, an isocyanate, an isothiocyanate, a N-hydroxysuccinimide ester, a carbodiimide, an anhydride, a sulfonyl chloride, an aldehyde, an oxirane, an acyl azide, a carbonate, an aryl halide, an imidoester, a glyoxal, or any combination thereof, and the functional group of the parasitic material comprises a primary amine.Methods For Analyzing Fecal Matter Using Fluorochromes That Covalently Bond To Parasitic Material Inventor: Slusarewicz et al Atty Dkt. No. PSGOO 13 WO- 144870-546. The method of claim 1, wherein the fluorochrome comprises a fhiorophore comprising an isothiocyanate functional group.

7. The method of claim 1, wherein the fluorochrome comprises fluorescein isothiocyanate of fluorescein NHS ester.

8. The method of claim 1, wherein the fluorochrome is dissolved in a water-miscible, aprotic solvent prior to staining.

9. The method of claim 8, wherein the water-miscible, aprotic solvent comprises dimethyl sulfoxide or acetonitrile.

10. The method of claim 1, further comprising treating the parasitic material with a buffer to modify the pH prior to or concurrent with labeling.

11. The method of claim 1, wherein the labeled parasitic material comprises protozoan oocysts.

12. The method of claim 11, wherein the labeled parasitic material further comprises ova.

13. The method of claim 1, wherein the parasitic material is not bleached prior to labeling.

14. The method of claim 1, wherein the flotation medium comprises an aqueous solution comprising sodium chloride, sodium nitrate, zinc sulfate, magnesium sulfate, sucrose, or a combination thereof.

15. The method of claim 1, wherein extracting at least a portion of the parasitic material from the flotation medium comprises pipetting the parasitic material from a surface of the flotation medium.

16. The method of claim 1, further comprising filtering non-parasitic material from the flotation medium with a filter having a pore size of from 10 to 1,000 microns.Methods For Analyzing Fecal Matter Using Fluorochromes That Covalently Bond To Parasitic Material Inventor: Slusarewicz et al Atty Dkt. No. PSGOO 13 WO- 144870-5417. The method of claim 1, wherein extracting at least a portion of the parasitic material from the flotation medium comprises filtering the parasitic material from the flotation medium using a capture filter.

18. The method of claim 17, wherein the capture filter has a pore size of from 5 to 50 microns.

19. The method of claim 1, further comprising washing excess fluorochrome from the parasitic material after labeling and before imaging.

20. The method of claim 1, wherein the functional group of the parasitic material comprises an amine, a thiol, a carboxylic acid, an alcohol, or an aldehyde.

Images