Patent Description:
Quantification of bacteria, yeast, and other organisms in fluid can be useful for medical diagnosis, drug development, industrial hygiene, food safety, and many other fields. For example, the amount of bacteria in urine is an important parameter in clinical diagnosis of an infection. In general, the presence of bacteria of <NUM><NUM>/mL or more in urine is recognized as a criterion of positive urinary tract infection. Urine samples having <NUM><NUM>/mL or fewer bacteria are considered negative for urinary tract infection (i.e., as having normal bacteria flora). If the urine sample has about <NUM><NUM>/mL bacteria, however, no diagnosis is established and the sample is often re-tested.

Urine often contains contaminants such as sediment, debris, mucus threads, crystals, amorphous salts, patient cells, red blood cells, and cell fragments. Some of the many contaminants are provided in <FIG>. These substances hinder the measurement of other types of particles (in particular bacteria) so that it has been difficult to accurately count the number of bacteria. Conventionally, observation of bacteria in urine has been performed by microscopic examination of stained bacteria. Bacterial staining also results in contaminants being stained simultaneously, and hinders the accurate measurement of the amount of bacteria.

<CIT> discloses methods for stabilizing and maintaining the viability of microorganisms from sample collection to downstream analysis comprising contacting the biological sample with a stabilization composition.

<CIT> a method of treating a viscous and particulate matter comprising sample with an extraction reagent comprising nitrous acid.

<CIT> discloses the selective clearing of a sample using deoxycholate as reagent.

Accordingly, there is a need for improved systems and methods that quickly determine whether bacteria are present in the fluid sample and determine the amount of bacteria. There is also a need for an improved systems and methods that more quickly determine the type of bacteria after it is determined that bacteria are present.

The present invention relates in one aspect to a method of measuring an amount of bacteria in a biological sample, the method comprising: clearing the sample of non-bacterial particulate matter by contacting the biological sample with an aqueous clearance solution comprising one or more surfactants comprising a fatty acid amide derivative of N-methyltaurine or a salt thereof, wherein the fatty acid is a monounsaturated or polyunsaturated C<NUM> - C<NUM> fatty acid; and determining the amount of bacteria in the sample.

In a further aspect, the invention relates to a method of clearing non-bacterial particulate matter from a biological sample, the method comprising: contacting the biological sample with an aqueous clearance solution one or more surfactants comprising a fatty acid amide derivative of N-methyltaurine or a salt thereof, wherein the fatty acid is a monounsaturated or polyunsaturated C<NUM> - C<NUM> fatty acid.

In a further aspect, the invention relates to a method of measuring an amount of bacteria in a biological sample, the method comprising:.

Further embodiments are detailed in the dependent claims.

The accompanying drawings are included to provide a further understanding of the methods and devices of the disclosure, and are incorporated in and constitute a part of this specification. The drawings are not necessarily to scale, and sizes of various elements may be distorted for clarity. The drawings illustrate one or more embodiment(s) of the disclosure, and together with the description, serve to explain the principles and operation of the disclosure.

The disclosure provides materials, methods, and apparatus to improve the measurement of an amount of bacteria in biological and other samples. For instance, the disclosure describes efficient, accurate, and cost-effective methods for minimizing the effect of the contaminants that can affect the accuracy of the measurement. For example, urine often contains contaminants, such as those shown in <FIG>, that interfere with the accurate measurement of the amount of bacteria. Accordingly, the method of the disclosure eliminates the interference caused by common urine contaminants (e.g., sediment, debris, mucus threads, crystals, amorphous salts, cell fragments, red blood cells, and patient cells) and allows for accurate measurement of bacteria. In a particular example of the methods of the disclosure include treating the sample to render the contaminants optically clear in the treated sample so that the accurate measurement of bacteria only can be accomplished. In addition, some embodiments of the methods of the disclosure are able to distinguish between two shapes of bacteria, rod and coccus, as well as cocci such as diplococci and cocci chains. Furthermore, in the methods of the disclosure, the bacteria in a biological sample may be measured using a single sample, a single image, and/or a single assay. As a result, the methods of the disclosure may eliminate multiple test methods, extensive sample handling, shipping, and storage. The measurements can be performed in-clinic and in real-time resulting in fewer changes to the sample.

As used herein, "measuring" or "measurement" of bacteria is not intended to be limiting and may involve determining the amount of bacteria (e.g., a total number of cells or a concentration), counting the bacteria, determining the size and/or shape of the bacteria, and/or determining the ability of the bacteria to be stained or labeled.

Thus, one aspect of the invention provides a method of measuring an amount of bacteria in a biological sample, the method comprising: clearing the sample of non-bacterial particulate matter by contacting the biological sample with an aqueous clearance solution comprising one or more surfactants comprising a fatty acid amide derivative of N-methyltaurine or a salt thereof, wherein the fatty acid is a monounsaturated or polyunsaturated C<NUM> - C<NUM> fatty acid; and determining the amount of bacteria in the sample.

Another aspect of the invention provides a method for clearing non-bacterial particulate matter from a biological sample. The non-bacterial particulate matter includes, but is not limited to, sediment, debris, mucus threads, crystals, amorphous salts, cell fragments, red blood cells, and patient cells. This method includes contacting the biological sample with an aqueous clearance solution comprising one or more surfactants comprising a fatty acid amide derivative of N-methyltaurine or a salt thereof, wherein the fatty acid is a monounsaturated or polyunsaturated C<NUM> - C<NUM> fatty acid. Such method, in certain embodiments, reduces ambiguous results in measuring bacteria in a biological sample.

As described above, the methods as described herein are carried out on a biological sample. In some embodiments of the methods of the disclosure, the sample is a urine sample. In some embodiments of the methods of the disclosure, the sample is a blood sample (whole blood, serum, or plasma).

In the methods of the disclosure, the biological sample is contacted with an aqueous clearance solution. Water is primarily used in the aqueous solutions of the disclosure (e.g., in the aqueous clearance solution or the aqueous chelator solution). However, in addition to water, aqueous solutions may include one or more organic solvents. In certain embodiments, the aqueous solution is a water solution. In certain embodiments, the aqueous solution comprises water and one or more organic solvents (such as alcohols, dimethyl sulfoxide, dimethylformamide, and acetone). The one or more organic solvents may be present at a concentration of <NUM> - <NUM>% volume/volume in the aqueous solution. One of skill in the art will be able to select suitable concentrations, based in part on the limits of solubility.

The aqueous clearance solution comprises one or more surfactants as used according to the claims. While the surfactant is generally described herein in the singular, a plurality of surfactants can be formulated together to provide the aqueous clearance solution of the disclosure. When two or more surfactants are used in the aqueous clearance solution, the relative amounts of the two can be varied based on the disclosure herein, depending on the performance desired. In certain embodiments, the weight ratio of a first surfactant to a second surfactant is in the range of <NUM>:<NUM> to <NUM>:<NUM>.

Based on the disclosure herein, the surfactant can be selected to provide the aqueous clearance solution with desirable performance in eliminating interference caused by common urine contaminants. Moreover, the surfactant can be selected to avoid (e.g., prevent or not cause) the formation of micelles that are visible by light microscopy under the conditions of the methods of the disclosure. Such micelles can cause interference and appear substantially similar to bacteria when observed by light microscopy. Thus, in certain embodiments, the surfactant can be selected to avoid the formation of micelles that are substantially similar to bacteria by light microscopy.

Various surfactants are known in the art and can suitably be used in the methods and compositions described herein. In certain embodiments a, the surfactant is a N-methyltaurine derivative (e.g., a fatty acid amide) or a salt thereof. Suitable derivatives of N-methyltaurine include compounds having one or more chemical structure features of, for example, methyl oleoyl taurate provided below, such as an unsaturated hydrocarbon chain and/or taurate head group (which may be further substituted). Examples of unsaturated hydrocarbon chains include, but are not limited to those derived from oleic acid, linoleic acid, α-linolenic acid, stearidonic acid, eicosapentaenoic acid, docosahexaenoic acid, γ-linolenic acid, dihomo-γ-linolenic acid, arachidonic acid, docosatetraenoic acid, palmitoleic acid, paullinic acid, gondoic acid, erucic acid, nervonic acid, mead acid, linolelaidic acid, vaccenic acid, elaidic acid, myristoleic acid, sapienic acid, petroselinic acid, docosenoic acid, or gadolenic acid.

For example, the N-methyltaurine derivative may be represented by the following general formula:
<CHM>
wherein M+ is a cation and R is a monounsaturated or polyunsaturated C<NUM>-C<NUM> hydrocarbyl.

For purposes of this disclosure, "hydrocarbyl" is defined to be a monovalent group containing carbon and hydrogen, which may be linear or branched. Monounsaturated hydrocarbyl is defined as a hydrocarbyl having one double bond or one triple bond. In preferred embodiment, monounsaturated hydrocarbyl is a hydrocarbyl having one double bond. Polyunsaturated hydrocarbyl is defined as a hydrocarbyl having two or more double bonds, triple bonds, or combinations thereof. Any double bond in the hydrocarbyl group may be in the cis or trans configuration. In a preferred embodiment, at least one of the one or more double bonds is in the cis configuration.

In certain embodiment, R is monounsaturated (e.g., having one double bond) C<NUM>-C<NUM> hydrocarbyl (e.g., C<NUM>-C<NUM> hydrocarbyl, C<NUM>-C<NUM> hydrocarbyl, C<NUM>-C<NUM> hydrocarbyl, C<NUM>-C<NUM> hydrocarbyl, C<NUM>-C<NUM> hydrocarbyl, C<NUM>-C<NUM> hydrocarbyl, C<NUM>-C<NUM> hydrocarbyl, C<NUM>-C<NUM> hydrocarbyl, C<NUM>-C<NUM> hydrocarbyl, C<NUM>-C<NUM> hydrocarbyl, C<NUM>-C<NUM> hydrocarbyl, C<NUM>-C<NUM> hydrocarbyl, C<NUM>-C<NUM> hydrocarbyl, or C<NUM>-C<NUM> hydrocarbyl). In certain embodiments, R is cis-monounsaturated C<NUM>-C<NUM> hydrocarbyl (e.g., C<NUM>-C<NUM> hydrocarbyl, C<NUM>-C<NUM> hydrocarbyl, C<NUM>-C<NUM> hydrocarbyl, C<NUM>-C<NUM> hydrocarbyl, C<NUM>-C<NUM> hydrocarbyl, C<NUM>-C<NUM> hydrocarbyl, C<NUM>-C<NUM> hydrocarbyl, C<NUM>-C<NUM> hydrocarbyl, C<NUM>-C<NUM> hydrocarbyl, C<NUM>-C<NUM> hydrocarbyl, C<NUM>-C<NUM> hydrocarbyl, C<NUM>-C<NUM> hydrocarbyl, C<NUM>-C<NUM> hydrocarbyl, or C<NUM>-C<NUM> hydrocarbyl).

M+ may be any suitable cation as provided herein. Examples include, but are not limited to, Na+, K+, Mg<NUM>+, Ca<NUM>+, and NH<NUM>+. In certain embodiments, M+ is Na+.

In certain embodiments as otherwise described herein, the surfactant is a salt of methyl oleoyl taurate. Sodium salt of methyl oleoyl taurate (sodium methyl oleoyl taurate or SMOT) has the following structure:
<CHM>.

The aqueous clearance solution of the disclosure, in certain embodiments, comprises sodium methyl oleoyl taurate or a derivative thereof. In certain embodiments, the aqueous clearance solution of the disclosure comprises sodium methyl oleoyl taurate.

Other non-limiting examples of suitable N-methyltaurine derivatives and their salts include: methyl linoleoyl taurate having the structure
<CHM>
methyl linolenoyl taurate having the structure
<CHM>
methyl docosenoyl taurate having the structure
<CHM>
methyl palmitoleoyl taurate having the structure
<CHM>.

In certain embodiments as otherwise described herein, the surfactant is selected from one or more of methyl oleoyl taurate, methyl linoleoyl taurate, methyl linolenoyl taurate, methyl docosenoyl taurate, methyl palmitoleoyl taurate, and salts thereof.

In certain embodiments, the aqueous clearance solution of the disclosure comprises one surfactant selected from methyl oleoyl taurate, methyl linoleoyl taurate, methyl linolenoyl taurate, methyl docosenoyl taurate, and methyl palmitoleoyl taurate, or a salt thereof.

In certain embodiments as otherwise described herein, the surfactant is a bile acid or a salt thereof. In certain embodiments, bile acids are selected from cholalic acid and its salts (cholates) (e.g., sodium cholate), deoxycholic acid and its salts (deoxycholates) (e.g., sodium deoxycholate), chenodeoxycholic acid and its salts (e.g., sodium chenodeoxycholate), lithocholic acid and its salts (e.g., sodium lithocholate), and ursodeoxycholic acid and its salts (e.g., sodium ursodeoxycholate).

The one or more surfactants may be present in the clearance solution described herein in a variety of amounts. In certain embodiments, the one or more surfactants is present in the clearance solution in a concentration in the range of about <NUM> % w/v to about <NUM> % w/v, based on the total volume of the clearance solution. In certain embodiments, the one or more surfactants is present in a concentration in the range of about <NUM> % w/v to about <NUM> % w/v, for example, in the range of <NUM> % w/v to <NUM> % w/v, or <NUM> % w/v to <NUM> % w/v, or <NUM> % w/v to <NUM> % w/v, or <NUM> % w/v to <NUM> % w/v, or <NUM> % w/v to <NUM> % w/v, or <NUM> % w/v to <NUM> % w/v, or <NUM> % w/v to <NUM> % w/v, based on the total volume of the clearance solution. In certain embodiments, the one or more surfactants is present in a concentration in the range of about <NUM> % w/v to about <NUM> % w/v, for example, in the range of <NUM> % w/v to about <NUM> % w/v, or <NUM> % w/v to about <NUM> % w/v, or <NUM> % w/v to <NUM> % w/v, or <NUM> % w/v to <NUM> % w/v, or <NUM> % w/v to <NUM> % w/v, or <NUM> % w/v to <NUM> % w/v, or <NUM> % w/v to <NUM> % w/v, based on the total volume of the clearance solution. In certain embodiments, the one or more surfactants is present in a concentration in the range of about <NUM> % w/v to about <NUM> % w/v, for example, in the range of <NUM> % w/v to <NUM> % w/v, or <NUM> % w/v to <NUM> % w/v, or <NUM> % w/v to <NUM> % w/v, or <NUM> % w/v to <NUM> % w/v, or <NUM> % w/v to <NUM> % w/v, or <NUM> % w/v to <NUM> % w/v, based on the total volume of the clearance solution. In certain embodiments, the one or more surfactants is present in a concentration in the range of about <NUM> % w/v to about <NUM> % w/v, for example, in the range of <NUM> % w/v to <NUM> % w/v, or <NUM> % w/v to <NUM> % w/v, or <NUM> % w/v to <NUM> % w/v, or <NUM> % w/v to <NUM> % w/v, or <NUM> % w/v to <NUM> % w/v, based on the total volume of the clearance solution. In certain embodiments, the one or more surfactants is present in a concentration in the range of about <NUM> % w/v to about <NUM> % w/v, for example, in the range of <NUM> % w/v to <NUM> % w/v, or <NUM> % w/v to <NUM> % w/v, or <NUM> % w/v to <NUM> % w/v, or <NUM> % w/v to <NUM> % w/v, or <NUM> % w/v to <NUM> % w/v, based on the total volume of the clearance solution. In certain embodiments, the one or more surfactants is present in a concentration in the range of about <NUM> % w/v to about <NUM> % w/v, for example, in the range of <NUM> % w/v to <NUM> % w/v, or <NUM> % w/v to <NUM> % w/v, or <NUM> % w/v to <NUM> % w/v, or <NUM> % w/v to <NUM> % w/v, or <NUM> % w/v to <NUM> % w/v, based on the total volume of the clearance solution. In certain embodiments, the one or more surfactants is present in a concentration in the range of about <NUM> % w/v to about <NUM> % w/v, for example, in the range of <NUM> % w/v to <NUM> % w/v, or <NUM> % w/v to <NUM> % w/v, or <NUM> % w/v to <NUM> % w/v, or <NUM> % w/v to <NUM> % w/v, based on the total volume of the clearance solution. In certain embodiments, the one or more surfactants is present in a concentration in the range of about <NUM> % w/v to about <NUM> % w/v, for example, in the range of <NUM> % w/v to <NUM> % w/v, or <NUM> % w/v to <NUM> % w/v, or <NUM> % w/v to <NUM> % w/v, or <NUM> % w/v to <NUM> % w/v, or <NUM> % w/v to <NUM> % w/v, based on the total volume of the clearance solution.

Of course, the concentration of the one or more surfactants in the sample, e.g., after the biological sample and the clearance solution are combined is lower, than the concentration of surfactant in the clearance solution. Thus, in some embodiments, the one or more surfactants may be present in the sample in a concentration in the range of about <NUM> % w/v to about <NUM> % w/v, based on the total combined volume of the biological sample and the clearance solution. For example, in certain embodiments, the one or more surfactants is present in a concentration in the range of about <NUM> % w/v to about <NUM> % w/v, or about <NUM> % w/v to about <NUM> % w/v, or about <NUM> % w/v to about <NUM> % w/v, or about <NUM> % w/v to about <NUM> % w/v, or about <NUM> % w/v to about <NUM> % w/v, or about <NUM> % w/v to about <NUM> % w/v, or about <NUM> % w/v to about <NUM> % w/v, or about <NUM> % w/v to about <NUM> % w/v, based on the total combined volume of the biological sample and the clearance solution. In certain embodiments, the one or more surfactants is present in a concentration in the range of about <NUM> % w/v to about <NUM> % w/v, or about <NUM> % w/v to about <NUM> % w/v, or about <NUM> % w/v to about <NUM> % w/v, or about <NUM> % w/v to about <NUM> % w/v, or about <NUM> % w/v to about <NUM> % w/v, or about <NUM> % w/v to about <NUM> % w/v, or about <NUM> % w/v to about <NUM> % w/v, or about <NUM> % w/v to about <NUM> % w/v, based on the total combined volume of the biological sample and the clearance solution. In some embodiments, the one or more surfactants may be present in the sample in a concentration in the range of about <NUM> % w/v to about <NUM> % w/v, based on the total combined volume of the biological sample, the clearance solution, and the chelator solution. For example, in certain embodiments, the surfactant is present in a concentration in the range of about <NUM> % w/v to about <NUM> % w/v, or about <NUM> % w/v to about <NUM> % w/v, or about <NUM> % w/v to about <NUM> % w/v, or about <NUM> % w/v to about <NUM> % w/v, or about <NUM> % w/v to about <NUM> % w/v, or about <NUM> % w/v to about <NUM> % w/v, based on the total combined volume of the biological sample, the clearance solution, and the chelator solution. in certain embodiments, the one or more surfactants is present in a concentration in the range of about <NUM> % w/v to about <NUM> % w/v, or about <NUM> % w/v to about <NUM> % w/v, or about <NUM> % w/v to about <NUM> % w/v, or about <NUM> % w/v to about <NUM> % w/v, or about <NUM> % w/v to about <NUM> % w/v, or about <NUM> % w/v to about <NUM> % w/v, or about <NUM> % w/v to about <NUM> % w/v, or about <NUM> % w/v to about <NUM> % w/v, or about <NUM> % w/v to about <NUM> % w/v, or about <NUM> % w/v to about <NUM> % w/v, based on the total combined volume of the biological sample, the clearance solution, and the chelator solution.

The clearance solution may be acidic or basic. In certain embodiments, the clearance solution is acidic; e.g., having a pH of less than <NUM>. For example, the clearance solution may have a pH of ≤ <NUM>, e.g., a pH of about <NUM> to about <NUM>, or about <NUM> to about <NUM>, or about <NUM> to about <NUM>. In certain embodiments, the clearance solution may have a pH of ≤ <NUM>, e.g., a pH of about <NUM> to about <NUM>, or about <NUM> to about <NUM>, or about <NUM> to about <NUM>. In certain embodiments, the clearance solution may even have a pH of ≤ <NUM>, for example a pH of about <NUM> to about <NUM>.

To obtain the clearance solution having the desired acidic pH, a buffer of suitable acidic pH may be included. Such buffer, for example, may include one or more acids. Thus, in certain embodiments, the clearance solution comprises an inorganoc acid, such as phosphoric acid, or an organic acid. Suitable organic acids include, but are not limited to, formic acid, ascorbic acid, carbonic acid, carboxylic acids, citric acid, and acetic acid. In some embodiments, the organic acid is a weak acid. The pKa of acids suitable for use in the methods of invention may range from <NUM> to <NUM>. In certain embodiments, the pKa is in the range of about <NUM> to <NUM>, or about <NUM> to <NUM>, or about <NUM> to <NUM>. In certain embodiments, the pKa is in the range of about <NUM> to <NUM>, or about <NUM> to <NUM>, or about <NUM> to <NUM>, or about <NUM> to <NUM>. In certain embodiments, the pKa is in the range of about <NUM> to <NUM>, or about <NUM> to <NUM>, or about <NUM> to <NUM>, or about <NUM> to <NUM>. In certain embodiments, the clearance solution comprises carboxylic acid. In certain embodiments, the clearance solution comprises citric acid or acetic acid. In certain embodiments, the clearance solution comprises citric acid. In certain embodiments, the clearance solution may have chelating properties. For example, the citric acid may also act as a chelator during contacting of the sample with the clearance solution. In certain embodiments, the clearance solution may comprise a suitable chelating agent (i.e., in addition to the organic acid or instead of the organic acid). The examples of chelating agents are described below.

The concentration of the acid (e.g., organic acid) in the clearance solution may be selected to achieve the desired pH of the clearance solution. In certain embodiments, the concentration of the acid is in the range of about <NUM> to <NUM>, for example, in the range of about <NUM> to <NUM>, or <NUM> to <NUM>, or <NUM> to <NUM>. In certain embodiments, the concentration of the acid is in the range of about <NUM> to <NUM>, for example, in the range of about <NUM> to <NUM>, or <NUM> to <NUM>, or <NUM> to <NUM>, or <NUM> to <NUM>, or <NUM> to <NUM>. In certain embodiments, the concentration of the acid is in the range of about <NUM> to <NUM>, for example, in the range of about <NUM> to <NUM>, or <NUM> to <NUM>, or <NUM> to <NUM>, or <NUM> to <NUM>. In certain embodiments, the concentration of the acid is in the range of about <NUM> to <NUM>, for example, in the range of about <NUM> to <NUM>, or <NUM> to <NUM>, or <NUM> to <NUM>. In certain embodiments, the concentration of the acid is in the range of about <NUM> to <NUM>, for example, in the range of about <NUM> to <NUM>, or <NUM> to <NUM>, or <NUM> to <NUM>, or <NUM> to <NUM>. In certain embodiments, the concentration of the acid is in the range of about <NUM> to <NUM>, for example, about <NUM> to <NUM>.

The concentration of the acid in the sample, e.g., after the biological sample and the clearance solution are combined, is lower than the concentration of the acid in the clearance solution. Thus, in some embodiments, acid may be present in the sample in a concentration in the range of about <NUM> to <NUM>, based on the total combined volume of the biological sample and the clearance solution. For example, the concentration of the acid is in the range of about <NUM> to <NUM>, or <NUM> to <NUM>, or <NUM> to <NUM>, or <NUM> to <NUM>, or <NUM> to <NUM>, based on the total combined volume of the biological sample and the clearance solution. In certain embodiments, the concentration of the acid is in the range of about <NUM> to <NUM>, for example, in the range of about <NUM> to <NUM>, or <NUM> to <NUM>, or <NUM> to <NUM>, or <NUM> to <NUM>, based on the total combined volume of the biological sample and the clearance solution. In certain embodiments, the concentration of the acid is in the range of about <NUM> to <NUM>, for example, in the range of about <NUM> to <NUM>, or <NUM> to <NUM>, based on the total combined volume of the biological sample and the clearance solution.

In certain embodiments, the clearance solution is basic; e.g., having a pH of more than <NUM>. For example, the clearance solution may have a pH ≤ <NUM>, e.g., a pH of about <NUM> to about <NUM>, or about <NUM> to about <NUM>, or about <NUM> to about <NUM>. In certain embodiments, the clearance solution may have a pH of ≥ <NUM>, e.g., a pH of about <NUM> to about <NUM>, or about <NUM> to about <NUM>, or about <NUM> to about <NUM>. To obtain such clearance solution having the desired basic pH, a buffer of suitable basic pH may be included. Suitable basic buffers and concentrations may be as described below with respect to the chelator solution.

In certain embodiments, the methods of the disclosure may further include contacting the sample with an aqueous chelator solution. In some embodiments, contacting with the chelator solution is performed after contacting with the clearance solution. In some embodiments, contacting with the chelator solution is performed before contacting with the clearance solution.

The chelator solution may comprise one or more chelating agents. The chelating agent is particularly useful for dissolving crystals, such as calcium and magnesiumcontaining crystals (oxalate and struvite) appearing in urine. Any kind of agent may be used as long as it is a decrystalizing agent. Some examples of the chelating agents include, but are not limited to ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid tetrasodium salt (EGTA), ethylenediaminetetraacetic acid tetrasodium salt dehydrate (EDTA), <NUM>,<NUM>-cyclohexylenedinitrilotetraacetic acid (CDTA), dihydroxyethylglycine (DHEG), diaminohydroxypropanetetraacetic acid (DPTA-OH), ethylenediamine-N,N'-diacetic acid (EDDA), ethylenediamine-N,N'-di-<NUM>-propionate (EDDP), glycoletherdiamine-N,N,N',N'tetraacetic acid (GEDTA), hydroxyethylethylenediaminetriacetic acid (HEDTA), lidofenin (HIDA), methyl-EDTA, trisodium nitrilotriacetate (NTA), pentetic acid, diethylenetriaminepentaacetic acid (DTPA), citric acid, sodium citrate, crown ethers, and the like. In certain embodiments, the chelator solution comprises ethylene glycol-bis(betaaminoethyl ether)-N,N,N',N'-tetraacetic acid tetrasodium salt (EGTA), ethylenediaminetetraacetic acid tetrasodium salt dehydrate (EDTA), or a combination thereof.

The concentration of the one or more chelating agents in the chelator solution may be selected to achieve the desired chelating activity. In certain embodiments, the concentration of each chelating agent in the chelator solution is in the range of about <NUM> to <NUM>, for example, in the range of about <NUM> to <NUM>, or <NUM> to <NUM>, or <NUM> to <NUM>, or <NUM> to <NUM>, or <NUM> to <NUM>. In certain embodiments, the concentration of each chelating agent is in the range of about <NUM> to <NUM>, for example, in the range of about <NUM> to <NUM>, or <NUM> to <NUM>, or <NUM> to <NUM>. In certain embodiments, the concentration of each chelating agent is in the range of about <NUM> to <NUM>, for example, in the range of about <NUM> to <NUM>, or <NUM> to <NUM>, or <NUM> to <NUM>, or <NUM> to <NUM>, or <NUM> to <NUM>. In certain embodiments, the concentration of each chelating agent is in the range of about <NUM> to <NUM>, for example, in the range of about <NUM> to <NUM>, or about <NUM> to <NUM>, or about <NUM> to <NUM>. In certain embodiments, the concentration of each chelating agent in the chelator solution is in the range of about <NUM> to <NUM>, for example, in the range of about <NUM> to <NUM>, or about <NUM> to <NUM>, or about <NUM> to <NUM>, or about <NUM> to <NUM>. In certain embodiments, the concentration of each chelating agent in the chelator solution is in the range of about <NUM> to <NUM>, for example, about <NUM> to <NUM>. In certain embodiments, the total concentration of the one or more chelating agents in the chelator solution is in the range of about <NUM> to <NUM>, for example, in the range of about <NUM> to <NUM>, or <NUM> to <NUM>, or <NUM> to <NUM>, or <NUM> to <NUM>, or <NUM> to <NUM>. In certain embodiments, the total concentration of the one or more chelating agents <NUM> to <NUM>, for example, in the range of about <NUM> to <NUM>, or <NUM> to <NUM>, or <NUM> to <NUM>, or <NUM> to <NUM>, or <NUM> to <NUM>. In certain embodiments, the total concentration of the one or more chelating agents is in the range of about <NUM> to <NUM>, for example, in the range of about <NUM> to <NUM>, or <NUM> to <NUM>, or <NUM> to <NUM>. In certain embodiments, the total concentration of the one or more chelating agents is in the range of about <NUM> to <NUM>, for example, in the range of about <NUM> to <NUM>, or <NUM> to <NUM>, or <NUM> to <NUM>, or <NUM> to <NUM>, or <NUM> to <NUM>. In certain embodiments, the total concentration of the one or more chelating agents is in the range of about <NUM> to <NUM>, for example, in the range of about <NUM> to <NUM>, or about <NUM> to <NUM>, or about <NUM> to <NUM>.

The concentration of the one or more chelating agents in the sample, e.g., after the biological sample, the clearance solution, and the chelator solution are combined, is lower than the concentration of the one or more chelating agents in the chelator solution. Thus, in some embodiments, the one or more chelating agents may be present in the sample in a concentration in the range of about <NUM> to <NUM>, based on the total combined volume of the biological sample, the clearance solution, and the chelator solution, for example, in the range of <NUM> to <NUM>, or <NUM> to <NUM>, or <NUM> to <NUM>, or <NUM> to <NUM>, or <NUM> to <NUM>, or <NUM> to <NUM>, or <NUM> to <NUM>. In certain embodiments, the one or more chelating agents has a concentration in the range of about <NUM> to <NUM>, for example, in the range of <NUM> to <NUM>, or <NUM> to <NUM>, or <NUM> to <NUM>, based on the total combined volume of the biological sample, the clearance solution, and the chelator solution. In certain embodiments, the one or more chelating agents has a concentration in the range of about <NUM> to <NUM>, for example, in the range of <NUM> to <NUM>, or <NUM> to <NUM>, or <NUM> to <NUM>, or <NUM> to <NUM>, based on the total combined volume of the biological sample, the clearance solution, and the chelator solution.

The chelator solution may be acidic or basic. In certain embodiments, the chelator solution is basic; e.g., having a pH of more than <NUM>. For example, the chelator solution may have a pH ≤ <NUM>, e.g., a pH of about <NUM> to about <NUM>, or about <NUM> to about <NUM>, or about <NUM> to about <NUM>. In certain embodiments, the chelator solution may have a pH of ≥ <NUM>, e.g., a pH of about <NUM> to about <NUM>, or about <NUM> to about <NUM>, or about <NUM> to about <NUM>.

To obtain the chelator solution having the desired basic pH, a buffer of suitable basic pH may be included. Such buffer, for example, may include one or more bases. Suitable bases include, but are not limited to, sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate, potassium carbonate, calcium carbonate, tris(hydroxymethyl)-aminomethane (TRIS), [tris(hydroxymethyl)methylamino]propanesulfonic acid (TAPS), <NUM>-(<NUM>-hydroxyethyl)-<NUM>-piperazineethanesulfonic acid (HEPES), <NUM>-[[<NUM>,<NUM>-dihydroxy-<NUM>-(hydroxymethyl)propan-<NUM>-yl]amino]ethanesulfonic acid (TES), <NUM>-(N-morpholino)propanesulfonic acid (MOPS), and the like. In certain embodiments, the chelator solution comprises a sodium hydroxide solution.

In certain embodiments, the chelator solution is acidic; e.g., having a pH of less than <NUM>. For example, the chelator solution may have a pH of ≤ <NUM>, e.g., a pH of about <NUM> to about <NUM>, or about <NUM> to about <NUM>, or about <NUM> to about <NUM>. In certain embodiments, the chelator solution may have a pH of ≤ <NUM>, e.g., a pH of about <NUM> to about <NUM>, or about <NUM> to about <NUM>, or about <NUM> to about <NUM>. In certain embodiments, the chelator solution may even have a pH of ≤ <NUM>. To obtain such chelator solution having the desired acidic pH, a buffer of suitable acidic pH may be included. Suitable acidic buffers and concentrations may be as described above with respect to the clearance solution.

The methods of the disclosure beneficially require less volume of sample than assay traditional methods for similar analytes. For instance, in certain embodiments of the methods of the disclosure, the sample volume is between about <NUM>µL and about <NUM>µL; for example, from about <NUM>µL to about <NUM>µL, or about <NUM>µL to about <NUM>µL, or about <NUM>µL to about <NUM>µL, or about <NUM>µL to about <NUM>µL, or about <NUM>µL to about <NUM>µL, or about <NUM>µL to about <NUM>µL, or about <NUM>µL to about <NUM>µL, or about <NUM>µL to about <NUM>µL.

The clearance solution of the disclosure may be added in a particular volume based on the desired applications. For example, in certain embodiments of the methods of the disclosure, the clearance solution volume is between about <NUM>µL and about <NUM>µL; for example, from about <NUM>µL to about <NUM>µL, or about <NUM>µL to about <NUM>µL, or about <NUM>µL to about <NUM>µL, or about <NUM>µL to about <NUM>µL, or about <NUM>µL to about <NUM>µL, or about <NUM>µL to about <NUM>µL, or about <NUM>µL to about <NUM>µL, or about <NUM>µL to about <NUM>µL, or about <NUM>µL to about <NUM>µL.

The chelator solution of the disclosure may be added in a particular volume based on the desired applications. For example, in certain embodiments of the methods of the disclosure, the chelator solution volume is between about <NUM>µL and about <NUM>µL; for example, from about <NUM>µL to about <NUM>µL, or about <NUM>µL to about <NUM>µL, or about <NUM>µL to about <NUM>µL, or about <NUM>µL to about <NUM>µL, or about <NUM>µL to about <NUM>µL, or about <NUM>µL to about <NUM>µL, or about <NUM>µL to about <NUM>µL, or about <NUM>µL to about <NUM>µL, or about <NUM>µL to about <NUM>µL.

In one example embodiment, the method of the disclosure as described herein includes: contacting the biological sample with an acidic aqueous clearance solution comprising one or more surfactants as defined in the claims, followed by contacting the sample with a basic aqueous chelator solution comprising one or more chelating agents. In another example embodiment, such method as described herein further includes determining the amount of bacteria in the sample.

In one example embodiment, the method of the disclosure as described herein includes: contacting the biological sample with a basic aqueous chelator solution comprising one or more chelating agents followed by contacting the sample with an acidic aqueous clearance solution comprising one or more surfactants as defined in the claims. In another example embodiment, such method as described herein further includes determining the amount of bacteria in the sample. In certain embodiments, the sample is centrifuged prior to determining the amount of the bacteria. In some embodiments, the centrifugation may be performed before or after addition of the clearance solution. In further embodiments, the centrifugation may be performed before or after addition of the chelator solution. In other embodiments, the sample is not centrifuged prior to determining the amount of the bacteria.

After the treatment with the clearance solution and chelator solution (if used) but prior to determining the amount of bacteria in the sample, the sample may be maintained undisturbed (i.e., incubated) for a period of time. Thus, in certain embodiments, the sample may be incubated for about <NUM> seconds to about <NUM> minutes. For example, the methods may include incubating the sample for about <NUM> seconds to about <NUM> minutes, or about <NUM> seconds to about <NUM> minutes, or about <NUM> seconds to about <NUM> minutes, or about <NUM> seconds to about <NUM> minutes, or about <NUM> minute to about <NUM> minutes, or about <NUM> minute to about <NUM> minutes, or about <NUM> minutes to about <NUM> minutes, or about <NUM> minute to about <NUM> minutes, or about <NUM> minute to about <NUM> minutes, or about <NUM> minutes to about <NUM> minutes, or about <NUM> minutes to about <NUM> minutes, or about <NUM> minutes to about <NUM> minutes, or about <NUM> minutes to about <NUM> minutes, or about <NUM> minutes to about <NUM> minutes, or about <NUM> minutes to about <NUM> minutes, or about <NUM> minutes to about <NUM> minutes, or about <NUM> minutes to about <NUM> minutes.

In certain embodiments, the methods of the disclosure may further include contacting the sample with a stain or dye for the bacteria. Some examples of the suitable stains and dyes include, but are not limited to, Wright stain (methylene blue optionally with eosin), crystal violet dye (e.g., with grams iodine and safranin), and antibodies tagged with fluorescent molecules. In some embodiments, the bacteria are premeabilized by prior to the contacting of the sample with a stain or dye. Of course, in certain embodiments, the amount of bacteria in the sample is determined without staining or dying the bacteria. In additional embodiments, the bacteria are not permeabilized.

The amount of bacteria may be determined by counting bacteria or by analyzing bacteria (e.g., for bacterial shape or size). Such methods may involve analysis of the sample image under a light microscope. In some embodiments, the sample may be applied to a microscope slide, a capillary or a cuvette, such as a SediVue® cuvette (IDEXX Laboratories Inc. , Westbrook, Maine, U. ) Numerous microscopy techniques may be used, such as bright-field, dark-field, and fluorescence microscopy. In some embodiments, counting may be performed manually (e.g., without the aid of computer imaging). For example, manual counting may be performed by a human viewing the sample directly through a microscope. Manual counting may involve counting the number of bacteria per grid area on a microscope slide or the like, and using this count to calculate the number of bacteria per volume unit of urine. In some embodiments, the bacteria may be analyzed under microscope to determine shape (e.g., coccus, including cocci such as diplococci and cocci chains, or rod) and/or determine size. In some embodiments, analyzing may be performed manually (e.g., without the aid of computer imaging). For example, manual analysis may be performed by a human viewing the sample directly through a microscope.

The amount of bacteria may also be determined by.

In certain embodiments, the predetermined number of pixels may be obtained by independently performing an empirical experiment using a standard sample having a known amount of bacteria.

In some embodiments, an algorithm, program, or software may be used to quantify the amount of bacteria in a sample. In some embodiments, a computing device coupled to or in communication with the camera executes instructions (e.g., instructions stored in a memory of the computing device) in order to measure an amount of bacteria in the sample. Such instructions may be executed by a processor of the computing device in order to automate a portion of the measurement described above in relation to naked-eye techniques. For example, the instructions may be configured to measure the amount of bacteria in the sample by measuring or determining a number of pixels that correlates to the bacteria (e.g., corresponds to bacteria cells or an area of the image that consists of a bacteria cells) in an image of the sample. Additionally or alternatively, the instructions may cause the computing device to measure an amount of bacteria in the sample by measuring an area of an image of the sample that includes bacteria. Measuring the area that includes the bacteria may include determining a number of pixels of an image that relate to the bacteria. Differentiating the bacteria from the background of the image may include analyzing a numerical value associated with the pixels. For example, each pixel in an image could include a value corresponding to an amount of collected light, a level of intensity, brightness, coloration, greyscale, or another optical property of the pixel (e.g., a pixel in <NUM>-bit image could be represented by a number between <NUM> and <NUM>, where <NUM> corresponds to black and <NUM> corresponds to white). In a particular example, a threshold value could be set such that pixels with a value higher than the threshold are considered as comprising the bacteria, while those with a value lower than the threshold value are considered background. In such a case, determining a number of pixels that relate to the bacteria may include determining a number of pixels that are above or below some threshold value.

As described above, the methods as described herein are carried out on a biological sample to, for example, measure bacteria. In general, the methods of the disclosure allow for a portion of the bacteria in the biological sample to remain viable after carrying out the methods of the disclosure, e.g., after contact with the clearance solution, the chelator solution, or both. Thus, in certain embodiments, at least a portion of the bacteria in the sample remains viable after contact with the clearance solution, the chelator solution, or both. For example, in certain embodiments, at least <NUM>% of the bacteria in the sample remains viable after carrying out the methods of the disclosure, e.g., <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>%-<NUM>%, <NUM>-<NUM>%. In certain embodiments, at least <NUM>% of the bacteria in the sample remains viable after carrying out the methods of the disclosure, e.g., <NUM>%-<NUM>%, <NUM>-<NUM>%, or <NUM>-<NUM>%. In certain embodiments, at least <NUM>% of the bacteria in the sample remain viable after carrying out the methods of the disclosure, e.g., <NUM>-<NUM>%, <NUM>-<NUM>%, or <NUM>-<NUM>%. In certain embodiments, essentially all of the bacteria (e.g., at least <NUM>%, <NUM>%, or even <NUM>%) in the sample remains viable after carrying out the methods of the disclosure.

In certain embodiments, less than <NUM>% of the bacteria in the biological sample remain viable after carrying out the methods of the disclosure; for example, less than <NUM>%, or less than <NUM>%, or essentially no bacteria remains viable after carrying out the methods of the disclosure.

In certain embodiments, the methods of the disclosure include first determining that the biological sample contains non-bacterial particulate matter that interferes with the analysis of for the bacteria, and then carrying out the methods as described herein (e.g., conacting the biological sample with the aqueous clearance solution, etc.). One of skill in the art recognizes that only a portion of the biological sample would be used in the first determination and that a different portion of the biological sample would be contacted with the aqueous clearance solution. Therefore, in certain embodiments, the methods of the disclosure further include, prior to contacting the sample with the clearance solution: withdrawing a portion of the biological sample, analyzing the portion for bacteria; and determining that the presence or amount of non-bacterial particulate matter interferes with the analysis for bacteria.

In certain embodiments, the invention provides methods for measuring bacteria in a biological sample, including:.

In some embodiments, the methods of the disclosure may further include making a determination about a health state of the patient based on at least the determined concentration of bacteria in the sample. In some cases, a computing device (e.g., a computer, a server, a processor, or a controller) in communication with the camera may use the determined concentration of bacteria to determine a health state, diagnose a disease (such as an infection), express a risk factor, or offer some other information about the health of a patient. In some embodiments, diagnosing a health state (e.g., a bacterial infection) may include determining whether the amount of bacteria is above or below a threshold value. Other health states and analyses are envisioned by one of ordinary skill in the art. Commonly, the health state is a urinary tract infection.

Throughout this specification, unless the context requires otherwise, the word "comprise" and "include" and variations (e.g., "comprises," "comprising," "includes," "including") will be understood to imply the inclusion of a stated component, feature, element, or step or group of components, features, elements or steps but not the exclusion of any other integer or step or group of integers or steps.

As used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise.

As used herein, "patient" refers to a warm blooded animal such as a mammal, preferably a human, cat, dog, rodent, primate, or horse. Patients may be healthy, or suffer from or have a potential to be afflicted with one or more diseases and disorders described herein. In some embodiments, the biological samples of the disclosure may be obtained or derived from patients.

As used herein, "salt' includes acid addition salts of compounds of the present disclosure. The salts may include cations based on the alkali and alkaline earth metals, such as sodium, lithium, potassium, calcium, magnesium, and the like, as well as ammonium, quaternary ammonium, and amine cations including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like.

The methods of the disclosure are illustrated further by the following examples.

Fresh, refrigerated urine was stored at <NUM>-<NUM> until shortly before analysis. The sample was allowed to warm to room temperature (approximately <NUM>) prior to analysis. Analysis should be performed as soon as possible following receipt of the sample (e.g., within <NUM> hours).

Clearance solution was prepared by dissolving <NUM> of sodium methyl oleoyl taurate (available as Geropon T77 from Solvay, <NPL>) in <NUM> of <NUM> citric acid. Chelator solution was made to have <NUM> ethylenediaminetetraacetic acid tetrasodium salt dehydrate (EDTA, available from Sigma; <NPL>), <NUM> ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid tetrasodium salt (EGTA, available from Sigma; <NPL>) in <NUM> sodium hydroxide.

Samples that are sufficiently crowded to make it difficult to identify bacteria and samples having small debris that is difficult to distinguish from bacteria are particularly well suited for this procedure.

In particular, <NUM>µL of well-mixed, unspun urine sample was placed into the appropriate flat bottom microtiter well. Then, <NUM>µL of clearance solution was added into the well and mixed gently by pipetting up and down <NUM> times, taking care not to introduce unnecessary bubbles. <NUM>µL of chelator solution was then added and mixed gently by pipetting up and down <NUM> times, taking care not to introduce unnecessary bubbles. The sample was then let to settle for <NUM>-<NUM> minutes undisturbed. An inverted light microscope with phase contrast filter, <NUM>×, and <NUM>× objectives was used to determine the amount of bacteria.

Blood-containing canine urine was treated using the materials and procedure described above. As provided in <FIG>, the before-treatment sample contained a significant amount of blood cells and other contaminants. After treatment, the sample was optically clear of the blood cells and other contaminants (<FIG>).

Canine whole blood and/or E. coli grown in LB broth were added to a medium of <NUM> filtered canine urine (referred to herein as urine filtrate). The dilutions were <NUM>× of a <NUM> hour E. coli culture. For the blood sample, the total dilution was about <NUM>×. Water was used as a dilution control and added at ratio of <NUM>:<NUM> to urine filtrate. Water was selected as a control because both clearance and chelator solutions are aqueous. The clearance and chelator solutions are prepared according to Example <NUM>, and the same procedure was followed.

As provided in <FIG>, the method of the disclosure disrupts the blood cell membranes, while leaving the bacteria intact. Here the treatment with the method of the disclosure showed absence of bacteria in blood only sample, and presence of bacteria in the samples with E. The water control showed the presence of bacteria even in blood only sample (i.e., was a false positive result). The images of the results are provided in <FIG>.

Canine urine containing large crystalline debris as well as numerous blood cells and bacteria was combined <NUM>:<NUM> with a <NUM>% w/v solution of SMOT and mixed (i.e., the final concentration of SMOT was <NUM>%). The resulting mixture was combined <NUM>:<NUM> with a solution containing <NUM> EDTA and <NUM> EGTA (i.e., the final concentration was <NUM> of each chelator) and mixed gently to produce the treated sample. The untreated urine and the treated sample were observed and photographed with a custom semi-automated microscope system. The exemplary images of the results are provided <FIG>. The method of the disclosure (i.e., treatment with SMOT and chelating solution) cleared the urine of the crystalline debris and the blood cells, but the bacteria remained visible.

Three test biological samples were prepared as follows. "Blood" sample was prepared by adding <NUM>µL of canine whole blood into <NUM> of filtered sterile canine urine; "E. coli" sample was prepared by adding <NUM>µL of lysogeny broth (LB) containing a <NUM>-hour E. coli culture into <NUM> of filtered sterile canine urine; and "Blood and E. coli" sample was prepared by adding <NUM>µL of canine whole blood and <NUM>µL of LB containing a <NUM>-hour E. coli culture into <NUM> of filtered sterile canine urine.

A <NUM>% w/v solution of SMOT and a <NUM> % w/v solution of sodium cholate in water (<NPL>) were also prepared in water. <NUM>µL of SMOT solution, <NUM>µL of cholate solution, or <NUM>µL of plain water were added to <NUM>µL of each of the three test biological samples to obtain the treated sample. Then, <NUM>µL of the treated sample was transferred into the SediVue® cartridge for analysis on SediVue Dx® Urine Sediment Analyzer (available from IDEXX Laboratories, Inc. , Westbrook, Maine).

The results are shown in <FIG>. The left-hand side panels of the graph show the number of bacteria (cocci and rods) as measured by the instrument, with the x-axis representing the average number of bacteria per high power field. The right-hand side panels of the graph show the number of red blood cells (RBC) measured by the instrument, with the x-axis representing the relative abundance of RBCs. In the control samples (water), the instrument reported bacteria in the blood-containing urine sample. Thus, the RBC led to false positive bacterial counts. After treatment of the samples with either SMOT or sodium cholate, the blood-containing urine sample had significantly reduced overall bacteria counts as compared to water-treated samples. Therefore, treatment method of the disclosure on a urine sample with SMOT or sodium cholate is effective in clearing RBCs and improves the accuracy of bacterial counting.

SMOT was saturated to obtain sodium methyl stearoyl taurate (i.e., <NUM>-[methyl(octadecanoyl)amino]ethanesulfonate sodium salt or SMST), as follows. <NUM> of sodium-N-methyl-N-oleyl taurate (<NUM> mmol), ammonium formate (<NUM>, <NUM> mmol, <NUM> eq), and <NUM>% Pd/C (<NUM>, <NUM> mmol) was suspended in methanol (<NUM>), under inert atmosphere (Argon). The reaction mixture was stirred under inert atmosphere at room temperature overnight. The mixture was filtered through celite, and the celite was washed with <NUM> × <NUM> ethyl acetate. The combined organics removed under rotary evaporation to provide the product (Sodium-N-Methyl-N-Stearoyl Taurate) as a yellowish oil. SMOT clearance solution was prepared to have <NUM> % w/v SMOT in <NUM> citric acid; SMST clearance solution was prepared to have <NUM> % w/v SMST in <NUM> citric acid; and chelating solution was prepared to have <NUM> EDTA, <NUM> EGTA in <NUM> NaOH. A canine urine sample (<NUM>µL) was first treated with <NUM>µL of a clearance solution (i.e., SMOT or SMST). The concentration of SMOT in SMOT-treated sample was <NUM> % w/v, whereas the concentration of SMST in SMST-treated sample was <NUM> % w/v. Following treatment of the urine sample with the clearance solution, <NUM>µL of chelating solution was added. The treated sample was transferred into the SediVue® cartridge for analysis on SediVue Dx® Urine Sediment Analyzer.

As provided in <FIG>, treatment method of the disclosure with SMOT resulted in optical clearing of the sample. Treatment with SMST, however, did not yield optical clearing of the sample.

Six different chelator solutions were prepared: <NUM> EDTA solution; <NUM> EGTA solution; <NUM> EDTA solution; <NUM> EGTA solution; <NUM> / <NUM> EDTA / EGTA; and <NUM> / <NUM> EDTA / EGTA.

Each chelator solution (<NUM>µL) was individually added to <NUM>µL of canine urine containing crystals. Thus, the final concentrations of the treated samples were: <NUM> EDTA; <NUM> EGTA; <NUM> EDTA; <NUM> EGTA; <NUM> / <NUM> EDTA / EGTA; and <NUM> / <NUM> EDTA / EGTA. <NUM>µL of the treated sample was then added to a SediVue® cartridge for analysis on SediVue Analyzer. Here, the number of calcium oxalate crystals and magnesium ammonium phosphate (struvite) crystals per HPF (high power field) was assessed.

The results of the automated crystal counts after the treatment of the crystalcontaining urine sample with chelator solutions is provided in <FIG>. Treatment with each chelator solution resulted in a reduction of both calcium oxalate and struvite crystal counts with greater reduction in crystal counts at higher chelating agent concentrations.

In order to determine the effect of incubation time on crystal counts, <NUM>µL of chelator solution (<NUM> EDTA; <NUM> EGTA; or <NUM> EDTA and <NUM> EGTA) was individually added to <NUM>µL of canine urine containing crystals and allowed to incubate for <NUM> minutes prior to analysis on the instrument. The results of struvite crystal count determination are shown in <FIG>. Treatment with each chelator solution followed by a <NUM>-minute incubation period resulted in a further reduction of the struvite crystal count.

<NUM>µL aliquots of <NUM> % w/v SMOT was combined with varying amounts (from <NUM>µL to <NUM>µL) of <NUM> citric acid or <NUM> acetic acid to generate clearance solutions of different acidity. <NUM>µL of each clearance solution was added with gentle mixing to <NUM>µL of canine urine having crystalline debris and bacteria. The pH of the resulting solution was measured, and <NUM>µL of this solution was transferred to a SediVue® cartridge and the total crystal count (CRY) was measured using SediVue Analyzer.

The pH values and the total crystals count (CRY) are plotted against increasing amounts of acids, and these results are provided in <FIG>. Addition of either citric acid or acetic acid resulted in a reduction of the CRY count. The CRY counts dropped to zero with the addition of at least <NUM>µL of citric acid (having final pH of <NUM>), or with the addition of at least <NUM>µL of acetic acid (having final pH of <NUM>). But as shown in <FIG>, acetic acid resulted in a sharp increase in bacterial count at pH values below <NUM>, suggesting that treatment with acetic acid results in features that are falsely counted as bacteria. Thus, in certain embodiments, citric acid may be used in the clearance solution at a wide range of pH values. In certain embodiments, acetic acid may be used in the clearance solution at pH of ≥<NUM>.

<NUM>µL canine urine having crystalline debris (mostly struvites) was treated with <NUM>µL of a series of clearance solutions containing <NUM> w/v % SMOT and ranging from <NUM> to <NUM> citric acid, followed by gentle mixing. <NUM>µL of this mixture was then combined with <NUM>µL of water and gently mixed. <NUM>µL of the resulting mixture was then placed into a SediVue® cartridge and the total crystal count was measured using SediVue Analyzer. As provided in Table <NUM>, the total crystal count per high power field decreased with increasing concentrations of citric acid in the clearance solution.

Next, the effect of the pH on the chelator solution on total crystal count was examined. <NUM>µL of the canine urine was combined with <NUM>µL of water, or with <NUM>µL of a clearance solution containing <NUM> % w/v SMOT in <NUM> citric acid, followed by gentle mixing. <NUM>µL of this mixture was then combined with <NUM>µL of a series of chelator solutions containing <NUM> EDTA, <NUM> EGTA, and NaOH (<NUM>, <NUM> or <NUM>), and mixed gently. <NUM>µL of the resulting mixture was then placed into a SediVue® cartridge and the total crystal count was measured using SediVue Analyzer. As provided in Table <NUM>, the total crystal count per high power field decreased with the addition of EDTA and EGTA. The total crystal count further decreased with increasing concentrations of NaOH (i.e., increasing pH) in the clearance solution.

<NUM>µL canine urine containing urinary crystals, bacteria, and <NUM> % v/v of canine blood was combined with <NUM>µL of clearance solution (<NUM> % w/v SMOT in <NUM> citric acid) and mixed gently. The final concentration of SMOT in the treated urine sample was <NUM> %w/v and citric acid was <NUM>. Then, to <NUM>µL of this mixture <NUM>µL of chelator solution (<NUM> EGTA, <NUM> EDTA, <NUM> NaOH) was added and mixed gently. The final concentration of EGTA and EDTA after the addition was <NUM> each and NaOH was <NUM>. <NUM>µL of the resulting mixture was then placed into a SediVue® cartridge and analyzed using SediVue Analyzer, either immediately or after a <NUM>-minute incubation. The negative control was the untreated canine urine containing urinary crystals, bacteria, and <NUM> % v/v of canine blood.

<FIG> shows microscopic images of the urine without treatment, with treatment and immediate analysis, and with treatment after a <NUM>-minute incubation. With immediate analysis, the vast majority of red blood cells (RBCs) and crystals had been cleared from the sample, but the bacteria remained visible. The four minute incubation yielded a further improvement in the clearing of the sample from crystals and cells debris, but the bacteria remained visible. For example, the number of RBCs per high power field, as counted by the instrument (without the <NUM>-minute incubation), was <NUM> in the untreated sample and <NUM> in the treated sample. Thus, the treatment method of the disclosure yielded a reduction in RBC count of <NUM>%. The number of total crystals per high power field (without the <NUM>-minute incubation), as counted by the instrument, was <NUM> in the untreated sample and <NUM> in the treated sample. Thus, the treatment method of the disclosure yielded a reduction in total crystal count of <NUM>%.

Next, <NUM>µL of highly crystallized canine urine known to contain bacteria was treated with <NUM>µL of clearance solution as described above and mixed gently. To <NUM>µL of this mixture, <NUM>µL of chelator solution as described above was added and mixed gently. An aliquot of the treated final sample was compared with the untreated highly crystallized canine urine using light microscopy. As shown in <FIG>, the urinary crystals and the bacteria were visible in the untreated urine. Only the bacteria, but not the crystals, were visible in the treated urine. Thus, the treatment method of the disclosure was able to dissolve the crystals in the highly crystallized samples. The treatment method cleared the urine of the crystals as determined by light microscopy, and maintained the bacteria intact and, for example, available for further analysis.

Another experiment was carried out as described above on <NUM>µL of canine urine containing mucus, cellular debris, and bacteria. Clearance solution and chelator solution were as described above. An aliquot of the treated sample was compared to the untreated urine by light microscopy. The mucus, cellular debris, and bacteria (including cocci chains) were visible in the untreated urine. In the treated urine, the bacteria, including cocci chains, were visible; the mucus and cellular debris were not visible. Thus, the treatment method of the disclosure cleared the urine of the mucus and cellular debris as determined by light microscopy, and maintained the bacteria intact (including undisrupted cocci chains).

Finally, the performance of bacterial recognition by the instrument before and after treatment was compared to quantitative culture. <NUM> fresh urine samples known to contain bacteria were used in this experiment. For quantitative culture, the urine samples were diluted and plated onto blood agar plates according to procedures well-known in the art. For example, the urine samples were diluted <NUM>:<NUM>, and <NUM>µL of the dilution was plated onto blood agar. Following overnight incubation, the number of colonies was counted and the titer of bacteria per milliliter urine was calculated. Samples were then grouped into positive/negative bins according to three thresholds: <NUM><NUM> cells/mL, <NUM><NUM> cells/mL, and <NUM><NUM> cells/mL. For example, at a threshold of <NUM><NUM> cells/mL, urine samples with <NUM><NUM> bacteria/mL or above were deemed positive, and urine samples with less than <NUM><NUM> bacteria/mL were deemed negative. For determination on the SediVue Analyzer, <NUM>µL of urine sample was combined with <NUM>µL of clearance solution as described above and mixed gently. To <NUM>µL of this mixture, <NUM>µL of chelator solution as described above was added and mixed gently. <NUM>µL of the resulting mixture was then placed into a SediVue® cartridge and analyzed using SediVue Analyzer. The Analyzer returned "positive" or "negative" calls. The results are provided in Table <NUM>.

In Table <NUM>, the calls made by the SediVue Analyzer were compared to the culture results. For example, at the <NUM><NUM> bacteria/mL concentration, <NUM> samples were negative by culture. Of these, <NUM> were called negative by the Analyzer with treatment and <NUM> were called positive by the Analyzer with treatment. At the <NUM><NUM> bacterial concentration, <NUM> samples were positive by culture. Of these, <NUM> were called negative by the instrument with treatment and <NUM> were called positive by the instrument with treatment. The performance of SediVue Analyzer calls as compared to bacterial culture, before and after treatment, was as follows: At the <NUM><NUM> threshold, accuracy improved from <NUM>% to <NUM>%, sensitivity increased from <NUM>% to <NUM>%, and specificity decreased from <NUM>% to <NUM>%. At the <NUM><NUM> threshold, accuracy increased from <NUM>% to <NUM>%, sensitivity increased from <NUM>% to <NUM>%, and specificity decreased from <NUM>% to <NUM>%. At the <NUM><NUM> threshold, accuracy increased from <NUM>% to <NUM>%, sensitivity increased from <NUM>% to <NUM>% and specificity decreased from <NUM>% to <NUM>%. These results indicate that the treatment of the method of the disclosure improved the performance of microscopic bacterial counting across a wide range of urinary bacterial titers. It is important to note that the blood agar method will only count viable bacteria (i.e., colony forming units), while the optical counting method on the instrument will also count nonviable bacteria.

The performance of bacterial recognition by SediVue Analyzer, before and after treatment according to the method of the disclosure, was compared to quantitative culture. <NUM> fresh canine urine samples were used. For quantitative culture, the urine samples were diluted and plated onto blood agar plates as described above. Urines with a titer at or above <NUM><NUM> bacteria/mL were deemed positive by culture, and below <NUM><NUM> bacteria/mL were deemed negative by culture.

For automated microscopy, <NUM>µL of canine urine was treated with <NUM>µL of clearance solution (<NUM> % w/v SMOT in <NUM> citric acid) and mixed gently. The final concentration of SMOT in the treated urine sample was <NUM> %w/v and citric acid was <NUM>. Then, to <NUM>µL of this mixture <NUM>µL of chelator solution (<NUM> EGTA, <NUM> EDTA, <NUM> NaOH) was added and mixed gently. The final concentration of EGTA and EDTA after the addition was <NUM> each and NaOH was <NUM>. <NUM>µL of the resulting mixture was then placed into a SediVue® cartridge and analyzed using SediVue Analyzer. The negative control was the untreated canine urine. The instrument used an algorithm that categorized bacterial count results into three bins: negative, suspected positive (i.e., ambiguous), and positive. Results are provided in Table <NUM>.

As provided in Table <NUM>, for the untreated (neat) urines, the SediVue algorithm called <NUM> samples (<NUM>%) ambiguous (±) for the presence of bacteria. With the treated urines, the number of ambiguous calls was reduced to <NUM> (<NUM>%). Therefore, the treatment of the methods of the disclosure reduced the number of ambiguous bacterial calls made by the instrument. The performance of SediVue calls as compared to bacterial culture was as follows: accuracy increased from <NUM>% to <NUM>%, sensitivity increased from <NUM>% to <NUM>% and specificity increased from <NUM>% to <NUM>%. In summary, the method of the disclosure using the clearance solution and chelator solution as described above improved the performance of microscopic bacterial counting.

The <NUM> previously untreated urines with ambiguous bacterial calls were then subjected to additional treatments. The <NUM> urines were treated with clearance solution and chelator solution as described above. Eight of the <NUM> ambiguous samples had been flagged for overcrowding. These eight samples were separately: A) diluted (between <NUM>-fold and <NUM>-fold) and re-analyzed on the SediVue Analyzer and B) diluted (between <NUM>-fold and <NUM>-fold) prior to treatment with the clearance solution and the chelator solution, and re-analyzed on SediVue Analyzer. The results of the analyses are shown in Table <NUM>. The number of ambiguous results fell from <NUM> (<NUM>%) without treatment, to <NUM> (<NUM>%) after treatment. Thus, dilution, or a combination of dilution and treatment can further reduce the number of ambiguous calls, as compared to treatment alone.

The previous experiment was carried out as described above on <NUM>µL of canine urine containing crystalline debris or on <NUM>µL of canine urine containing crystalline debris and mucus. Clearance solution (<NUM>µL) and chelator solution (<NUM>µL) were as described above. Manual light microscopy of the resulting final mixture revealed that the treatment cleared crystalline debris from the urine sample containing crystalline debris. From the urine sample containing crystalline debris and mucus, manual light microscopy of the resulting final mixture revealed that the treatment cleared both the mucus and the crystalline debris.

To three filtered canine urine samples, <NUM> % v/v of blood was added to obtain blood-containing urine samples. These blood-containing urine samples (<NUM>µL) were each treated with <NUM>µL of clearance solution (SMOT at <NUM>% w/v, <NUM>% w/v, <NUM>% w/v or <NUM>% w/v, and <NUM> citric acid), and mixed gently. The final concentration of SMOT in the treated urine sample was <NUM> % w/v, <NUM> % w/v, <NUM> % w/v, or <NUM>% w/v. To <NUM>µL of the treated sample, <NUM>µL of chelator solution (<NUM> EDTA, <NUM> EGTA, and <NUM> NaOH) was added, followed by gentle mixing. Manual light microscopy of the resulting final mixtures revealed that all three urines were cleared of all crystals, debris and RBCs. Manual light microscopy of the resulting final mixtures revealed that final SMOT concentrations of <NUM> % w/v and above were sufficient to clear the RBCs from the urine sample.

Next, to five canine urine samples heavily crowded with crystals and debris, <NUM> % v/v of blood was added to obtain blood-containing urine samples. To <NUM>µL of each these blood-containing urine samples, <NUM>µL of clearance solution (<NUM> citric acid and SMOT ranging from <NUM> % w/v to <NUM> %w/v in increments of <NUM>%) was added, and mixed gently. The final concentration of SMOT in the treated urine sample ranged from <NUM> % w/v to <NUM> % w/v in increments of <NUM>%. To <NUM>µL of the treated sample, <NUM>µL of chelator solution (<NUM> EDTA, <NUM> EGTA and <NUM> NaOH) was added, followed by gentle mixing. Manual light microscopy of the resulting final mixtures revealed that, depending on the urine sample, the urine was cleared of crystals, debris and RBCs at final SMOT concentrations of: <NUM> % w/v and above, <NUM> % w/v and above, <NUM> % w/v and above, <NUM> % w/v and above, and <NUM> % w/v and above. Therefore, the final concentration of SMOT required to clear a heavily crowded urine sample from crystals, debris and RBCs can range from <NUM> % w/v to <NUM><NUM>% w/v.

In a further experiment, to canine urine samples known to be heavily crowded with crystals and debris, <NUM> % v/v blood was added. To each <NUM>µL blood-containing urine, <NUM>µL of a clearance solution (<NUM> % w/v SMOT and <NUM> citric acid) was added, and mixed gently. To <NUM>µL of the treated sample, <NUM>µL of chelator solution (<NUM> EDTA, <NUM> EGTA and <NUM> NaOH) was added, followed by gentle mixing. Manual light microscopy of the resulting final mixtures revealed that the sample urines were cleared of all crystals, debris and RBCs.

In a final experiment, to canine urine samples heavily crowded with crystals and debris, <NUM> % v/v blood was added. To each <NUM>µL blood-containing urine, <NUM>µL of a clearance solution (<NUM> % w/v SMOT and <NUM> citrate buffer with pH <NUM>) was added, and mixed gently. To <NUM>µL of the treated sample, <NUM>µL of chelator solution (<NUM> EDTA and <NUM> NaOH) was added, followed by gentle mixing. Manual light microscopy of the resulting final mixtures revealed that the sample urines were cleared of all crystals, debris and RBCs.

<NUM>µL of feline urine crowded with crystals, RBCs and white blood cells was combined with <NUM>µL of clearance solution (<NUM> % w/v SMOT in <NUM> citric acid) and gently mixed. Then, to <NUM>µL of this treated sample, <NUM>µL of chelator solution (<NUM> EGTA, <NUM> EDTA, <NUM> NaOH) was added and mixed gently. The resulting final mixture was observed by light microscopy.

Claim 1:
A method of measuring an amount of bacteria in a biological sample, the method comprising:
clearing the sample of non-bacterial particulate matter by contacting the biological sample with an aqueous clearance solution comprising one or more surfactants comprising a fatty acid amide derivative of N-methyltaurine or a salt thereof wherein the fatty acid is a monounsaturated or polyunsaturated C<NUM> - C<NUM> fatty acid; and
determining the amount of bacteria in the sample.