WO2012176202A1 - Articles and methods for monitoring urinary tract infection - Google Patents

Articles and methods for monitoring urinary tract infection Download PDF

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Publication number
WO2012176202A1
WO2012176202A1 PCT/IL2012/050209 IL2012050209W WO2012176202A1 WO 2012176202 A1 WO2012176202 A1 WO 2012176202A1 IL 2012050209 W IL2012050209 W IL 2012050209W WO 2012176202 A1 WO2012176202 A1 WO 2012176202A1
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WO
WIPO (PCT)
Prior art keywords
indicator composition
acid
indicator
another embodiment
range
Prior art date
Application number
PCT/IL2012/050209
Other languages
French (fr)
Inventor
Vered Pardo-Yissar
Yifat HERSHCOVITZ-FUNDOIANO
Hadar KESSARY SHOHAM
Original Assignee
Common Sense Ltd.
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Publication of WO2012176202A1 publication Critical patent/WO2012176202A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N31/00—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods
    • G01N31/22—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods using chemical indicators
    • G01N31/227—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods using chemical indicators for nitrates or nitrites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors

Definitions

  • the present invention relates to the field of medical diagnostics and more specifically, to an improved identification of urinary tract infection (UTI), using a urine-testing article comprising nitrite-indicator polymer matrix to identify nitrites in urine.
  • UTI urinary tract infection
  • the present invention further describes methods for preparing and using said urine-testing article.
  • Urinary tract infections are most common among females, with a high recurrence rate of about 25%-30%. UTI are considered the most frequent bacterial infections in women and it is estimated that about one third of all adult women have experienced an episode of symptomatic cystitis (bladder infection) at least once. UTI are common in infants and among young boys and young girls, albeit with considerably lower estimates. Most episodes of UTI are caused by bacterial infection; however in many cases the etiology is not clear. Typically, diagnosis of UTI includes dipping a dipstick in a urine sample for identifying elements, such as, leukocyte esterase or nitrites, which are indicative of infection.
  • identifying elements such as, leukocyte esterase or nitrites
  • Common diagnosis also includes examining proliferation of microorganisms in a urine culture.
  • the presence of nitrites in urine is a well-known marker for UTI, as the majority of bacteria that cause UTI induce reduction of nitrates to nitrites, thus causing a substantial elevation in nitrite level in the subject's urine.
  • U.S. Patent No. 4,631,255 discloses a composition for assaying for nitrites comprising an acid component, a diazotizing agent and a coupling agent, wherein said composition changes color upon contacting a urine sample containing nitrites.
  • Most diagnostic tools for UTI require the involvement of health care professionals, and therefore are used only in medical facilities. There are, however, diagnostic tools for UTI that are suitable for personal use at home.
  • 6,203,496 discloses a disposable article, such as, a diaper, a panty liner and a pad, for UTI preliminary diagnosis even by personnel having limited medical training.
  • the article comprises one or more chemical reagents that change color upon contact with urine containing abnormal levels of substances, including nitrites.
  • Suitable reagents include naphthyl ester, diazonium salt, arsenilic acid and tetrahydrobenzo (h) quinolin.
  • U.S. Patent No. 6,464,635 discloses a diaper comprising chemicals for visual examination of urine status, other than UTI, based on the level of various substances such as nitrates, bilirubin, ketones, blood, proteins, urobilinogen and glucose.
  • a secretion-monitoring article for identifying a secreted biological fluid, other than urine, providing a stable visual indication is disclosed in U.S. Patent No. 6,921,647.
  • the article comprises a pH indicator agent, a polymer and an ion-balance reagent where the indication is stable for at least 48 hours.
  • the present invention provides indicator compositions, diagnostic articles and uses thereof for detecting the presence of nitrites in a subject's urine, thereby diagnosing urinary tract infections.
  • the invention relies on the unexpected discovery that incorporating a chemically-based detection system for nitrites with a polymer provides a stable color indication.
  • the resulting indication does not leach and remains indicative for at least 24 hours.
  • the color indication is maintained even after several cycles of drying and re- wetting of the indicator system.
  • the indication provided by the article of the invention is not affected by interfering substances and conditions, such as glucose, ascorbic acid and changes in urine pH.
  • the present invention provides an indicator composition comprising a polymer, a plasticizer, a diazotizing reagent and a coupling agent, wherein said indicator composition provides a color indication in the presence of nitrites in urine.
  • the present invention provides a diagnostic article for detection of urinary tract infection in a subject, comprising an absorbent substrate configured to absorb urine and an indicator composition comprising a polymer, a plasticizer, a diazotizing reagent and a coupling agent, wherein said indicator composition provides a color indication in the presence of nitrites in said absorbed urine.
  • the present invention provides a diagnostic article for detection of urinary tract infection in a subject, comprising an absorbent substrate configured to absorb urine and the indicator composition of the invention, wherein said indicator composition provides a color indication in the presence of nitrites in said absorbed urine.
  • the indicator composition further comprises at least one component selected from the group consisting of: a pH indicator, a dye, an anti-oxidant, an acid, a trap, a photo-resistant agent and a wetting agent.
  • a solvent is added in order to obtain a homogenous solution of all the components in the composition.
  • the solvent may contain a buffer.
  • the buffer maintains an acidic pH.
  • the buffer maintains a transition pH range of 2.0-5.0.
  • the solvent is evaporated upon applying the indicator composition onto an absorbent substrate (thereby forming the article of the invention).
  • the amount of the polymer in the indicator composition is within the range of 30% to 90%, the amount of the plasticizer in the indicator composition is within the range of 5% to 50%, the amount of the diazotizing reagent in the indicator composition is within the range of 0.1% to 12%, the amount of the coupling agent in the indicator composition is within the range of 0.01% to 5%, wherein the percentages presenting the amount of each component in the indicator composition refer to weight/weight percentages based on the total weight of the indicator composition and the total weight of the indicator composition equals 100%.
  • the indicator composition further comprises pH indicator wherein the amount of the pH indicator in the indicator composition is within the range of 0.0005% to 1%.
  • the indicator composition further comprises a dye wherein the amount of the dye in the indicator composition is within the range of 0.05% to 2.5%.
  • the indicator composition further comprises an anti-oxidant, wherein the amount of the anti-oxidant in the indicator composition is within the range of 1% to 20%.
  • the indicator composition further comprises an acid, wherein the amount of the acid in the indicator composition is within the range of 0.1%- 10%.
  • the indicator composition further comprises a trap, wherein the amount of the trap in the indicator composition is within the range of 0.5% to 2.5%.
  • the indicator composition further comprises a wetting-agent, wherein the amount of the wetting agent in the indicator composition is within the range of 1% to 25%.
  • the indicator composition further comprises a photo-resistant agent, wherein the amount of the photo-resistant agent in said indicator composition is within the range of 0.1 % to 1 %.
  • the sensitivity of the indicator composition to nitrites is in the range of about 0.01 to 2 mg/dL.
  • said polymer comprises one or more of the polymers selected from the group consisting of: cellulose acetate, dextran, nitrocellulose, ethyl cellulose, carboxymethyl cellulose, cellulose acetate phthalate, cellulose acetate propionate, cellulose acetate butyrate, hydroxypropyl methylcellulose (HPMC), polyethylene glycol (PEG), polyvinyl alcohol, polyvinyl alcohol-polyethylene glycol graft copolymer, silicone dioxide and derivatives thereof.
  • said polymer is cellulose acetate.
  • said plasticizer is selected from the group consisting of: 1 ,2-cyclohexanedicarboxylic acid di-isononyl ester; bis(2-ethylhexyl) sebacate, acetyl tributyl citrate, triethylhexyl trimellitate, triethyl citrate, ethyl lactate, alkylsulfonic acid phenylester, dioctylphthalate, polylactide, ethyl hexyl phthalate, polyvinyl pyrrolidone (povidone) and combinations thereof.
  • 1 ,2-cyclohexanedicarboxylic acid di-isononyl ester bis(2-ethylhexyl) sebacate, acetyl tributyl citrate, triethylhexyl trimellitate, triethyl citrate, ethyl lactate, alkylsulfonic acid pheny
  • said plasticizer is 1 ,2-cyclohexane dicarboxylic acid di-isononyl ester.
  • said diazotizing agent is selected from the group consisting of: p-arsanilic acid, methyl anthranilate, sulfanilamide, sulfanilic acid, 2- amino-6-methoxy benzothiazole, 2-amino- benzothiazole, 2-amino-6-nitro benzothiazole, 2- amino-6-ethoxybenzothiazole, 2-amino-6-methylbenzothiazole, 2-amino-3,5- dinitrothiophene, 4-[(4-methyl-l-piperazinyl)sulfonyl]aniline, 4-( ⁇ 4'-[(4- aminophenyl)sulfonyl] [1,1 '-biphenyl]-4-yl ⁇ sulfonyl)aniline, Sulfadimethoxine, s
  • said diazotizing agent is sulfanilamide.
  • said diazotizing agent is 4-[(4-methyl-l- piperazinyl)sulfonyl]aniline.
  • said diazotizing agent is 4-( ⁇ 4'-[(4- aminophenyl)sulfonyl] [1,1 '-biphenyl]-4-yl ⁇ sulfonyl)aniline.
  • said coupling agent is selected from the group consisting of: N-(1-Naphthyl)ethylenediamine dihydrochloride, 4-( ⁇ 4'-[(4- aminophenyl)sulfonyl] [1,1 '-biphenyl] -4-yl ⁇ sulfonyl)aniline, 4- [(4-methyl- 1 - piperazinyl)sulfonyl] aniline, 1-Naphthylamine and 2-(l-Naphthylamino) ethanol.
  • the pH indicator has a transition pH range between 0-4.0.
  • the pH indicator with a transition range between 0-4.0 is selected from: leucomalachite green and gentian violet.
  • the pH indicator has a transition pH range between 8.0-14.0.
  • said dye is selected from the group consisting of: Indigo (2,2'-Bis(2,3-dihydro-3- oxoindolyliden)), Trypan Blue ((3 ⁇ ,3' ⁇ )-3,3'-[(3,3'- dimethylbiphenyl-4,4'-diyl)di( 1 Z)hydrazin-2-yl- 1 -ylidene]bis(5 -amino-4-oxo-3 ,4- dihydronaphthalene-2,7-disulfonic acid), Food Blue No.
  • Alizarin Blue Black B (disodium 4,4'-[(4-hydroxy-9,10-dioxo-9,10-dihydroanthracene-l,3- diyl)diimino]dibenzenesulfonate or other food or cosmetic grade color.
  • said dye is Indigo, also known as (2,2'-bis(2,3- dihydro-3- oxoindolyliden).
  • said anti-oxidant is selected from the group consisting of titanium dioxide pigments, zinc oxide, polyvinyl alcohol, ascorbic acid, D-Isoascorbic acid, t-butyl hydroquinone, polystyrene and vitamin E.
  • said trap is selected from the group consisting of: beta-cyclodextrin and pentasodium pentetate.
  • said wetting agent is selected from the group consisting of: glycerol, 2-ethoxy ethanol and sodium carboxy methyl starch.
  • said wetting agent is glycerol.
  • said acid is selected from the group consisting of: citric acid, tartaric acid, adipic acid, picric acid, methacrylic acid, stearic acid and acetic acid.
  • said acid is a salt derivative of an acid selected from the group consisting of: citric acid, tartaric acid, adipic acid, picric acid and acetic acid.
  • said acid is citric acid.
  • the photo-resistant agent is 4-methacryloxy-2- hydroxy benzo- phenone.
  • said indicator composition provides an indication which is stable, does not leach through several cycles of drying and re-wetting and is indicative for at least 3 hours upon contact with nitrites in urine, preferably for at least 12 hours, preferably for at least 24 hours and preferably for at least 48 hours.
  • the indication is stable for at least 3 hours.
  • the indication is stable for at least 12 hours.
  • the indication is stable for at least 24 hours.
  • the indication is stable for at least 48 hours.
  • the absorbent substrate is selected from the group consisting of: gauze, panty shield, hygienic napkin, diaper,inter-labial absorbent structure, an absorbent sticker and a combination thereof.
  • the absorbent substrate comprises a fabric material.
  • the fabric material is selected from the group consisting of: woven fabric, non- woven fabric, hydrophilic fabric material, hydrophobic fabric material and combinations thereof.
  • the diagnostic article further comprises a protective film separating the indication composition from the environment.
  • the protective film is configured to dissolve upon contacting urine.
  • the protective film comprises a polymer selected from the group consisting of: polyvinyl alcohol, polyethylene glycol, hydrous magnesium silicate, hydroxypropyl methyl cellulose, sodium starch glycollate, Polyethylene glycol (PEG), xanthan gum and a combination thereof.
  • the protective film comprises talc.
  • the present invention provides a method for detecting urinary tract infection in a subject, the method comprising
  • a diagnostic article comprising an absorbent substrate configured to absorb urine and an indicator composition
  • said indicator composition comprises a polymer, a plasticizer, a diazotizing reagent and a coupling agent and wherein said indicator composition provides a color indication in the presence of nitrites in said absorbed urine
  • detecting comprises observing the color indication of the indicator composition, wherein a color that is different from the color of said indicator composition prior to the step of mounting, is indicative of abnormal level of nitrites.
  • contacting the indicator composition with urine takes place upon receiving urine secreted from said subject.
  • the diagnostic article comprises a protective film wherein step (c) further comprises removing the protective film.
  • the present invention provides a method for preparing the article of the invention, the method comprising preparing a solution comprising the indicator composition and a solvent.
  • said solvent is selected from the group consisting of: water, isopropyl alcohol, ethanol, acetone and a combination thereof.
  • the present invention provides an indicator composition for detecting nitrites, a diagnostic article comprising the indicator composition and a method of using same to detect urinary tract infections in a subject in need thereof.
  • the article and methods of the invention are configured to be used by the user himself or a care taker, and do not require the involvement of personnel with medical training. Furthermore, the article and method of the invention may be applied outside medical facilities, providing theretofore outstanding patient compliance together with stable and reliable indication of UTI.
  • a main advantage of the indicator composition of the invention is the stable indication that it provides. The indication is maintained through cycles of drying and wetting, and is not affected by interfering substances and conditions, such as, glucose, and/or ascorbic acid and/or changes in urine pH. Without being bound to any theory or mechanism, these outstanding qualities are conferred by the specific combination of components in the indicator composition, including the presence of a polymer and a trap among other components.
  • the invention provides an indicator composition for detection of urinary tract infection in urine of a subject, the indicator composition comprising a polymer, a plasticizer, a diazotizing reagent and a coupling agent; wherein the indicator composition provides a color reaction in the presence of nitrites in the urine of said subject.
  • the color reaction provided by the indicator composition results in s color indication.
  • the terms "color reaction” and "color indication” are used interchangeably.
  • the invention provides a diagnostic article for detection of urinary tract infection in a subject, wherein the diagnostic article comprises an absorbent substrate configured to absorb urine and the indicator composition of the invention.
  • the indicator composition comprises a polymer, a plasticizer, a diazotizing reagent and a coupling agent, wherein the indicator composition provides a color reaction in the presence of nitrites in the absorbed urine.
  • the terms "urinary tract infection” and "UTI” are interchangeably used herein to define an infection of the urinary tract typically induced by bacteria.
  • the bacteria may be Escherichia coli and Staphylococcus saprophyticus among others.
  • the microorganisms that cause UTI induce reduction of nitrates to nitrites.
  • the level of nitrites in the infected urine is elevated.
  • nitrites give an indication of UTI.
  • the color indication provided by the indicator composition of the present invention is based on a chemical reaction between nitrites, in the urine, and the indicator composition.
  • the chemical reaction occurs between the nitrites and the diazotizing agent, followed by a coupling reaction, which produces an azo dye.
  • the color of the azo dye is thereby indicative of the presence of nitrites.
  • This type of chemical reaction is based on the Griess reaction which has been described in the art (Jay B. et al., Analytical Chemistry 1979, 51(9): 1493- 1502).
  • the color resulting from the reaction with the indicator-composition is stable.
  • the stable color indication provided by the indicator composition of the invention is distinctive from the color of urine, excrement or blood.
  • the color indication is of colors other than brown, red, or yellow.
  • the color indication is dependent on the combination of diazotizing reagent and coupling agent.
  • the color of the color indication is selected from the group consisting of: blue, green, orange, pink, purple, violet and white.
  • the color of the indication is determined by the diazotizing agent and the coupling agent or their combination.
  • the article of the invention may combine several indications corresponding to different physiological conditions.
  • the article may further include an indication for vaginosis.
  • the indicator disclosed in U.S. Pat. No. 6,627,394 may be incorporated in the article of the invention.
  • the variety of colors provided by the various diazotizing agents and coupling agents and their combination enable choosing distinct colors for each medical condition diagnosed by the same article.
  • stable color “stable color indication” and “stable indication” as used herein, are interchangeable. These terms refer to a color indication which maintains its color, or essentially the same color, over time.
  • the stable color indication is a color indication that does not leach through several cycles of drying and re- wetting. According to another embodiment, the stable color indication is a color indication that is indicative for at least 3 hours after contact with nitrites in urine. According to another embodiment, the stable color indication is a color indication that is indicative for at least 12 hours following contact with nitrites in urine. According to another embodiment, the stable color indication is a color indication that is indicative for at least 24 hours following contact with nitrites in urine. According to another embodiment, the stable color indication is a color indication that is indicative for at least 48 hours after contact with nitrites in urine.
  • the stable color indication is an indication which remains of essentially the same color, and does not leach or fade through several cycles of drying and re- wetting, for at least 3 hours upon contact with nitrites in urine or a combination thereof.
  • the polymer of the indicator-composition enables the stable color indication.
  • the plasticizer changes the plasticity of the polymer.
  • the amount of the polymer in the indicator composition is within the range of 30% to 90%, the amount of the plasticizer in the indicator composition is within the range of 5% to 50%, the amount of the diazotizing reagent in the indicator composition is within the range of 0.1% to 12% and the amount of the coupling agent in the indicator composition is within the range of 0.01% to 5%.
  • the percentages presenting the amount of each component in the indicator composition refer to weight/weight percentages based on the total weight of the indicator composition. The total weight of the indicator composition equals 100%.
  • the amount of plasticizer in the indicator composition is within the range of 15% to 50%.
  • the density of the plasticizer and wetting agent solutions is within the range of 0.6-1.26 gr/ml. According to yet another embodiment, the density of the plasticizer and wetting agent solutions is within the range of 0.3-1.5 gr/ml. According to another embodiment, the amount of the polymer in the indicator composition is within the range of 30% to 90%, the amount of the plasticizer in the indicator composition is within the range of 5% to 50%, the amount of the diazotizing reagent in the indicator composition is within the range of 0.1% to 28%, the amount of the coupling agent in the indicator composition is within the range of 0.01% to 5%, wherein the percentages presenting the amount of each component in the indicator composition refer to weight/weight percentages based on the total weight of the indicator composition and the total weight of the indicator composition equals 100%.
  • the amount of the polymer in the indicator composition is within the range of 30% to 90%
  • the amount of the plasticizer in the indicator composition is within the range of 15% to 50%
  • the amount of the diazotizing reagent in the indicator composition is within the range of 0.1% to 12%
  • the amount of the coupling agent in the indicator composition is within the range of 0.01% to 5%
  • the percentages presenting the amount of each component in the indicator composition refer to weight/weight percentages based on the total weight of the indicator composition and the total weight of the indicator composition equals 100%.
  • the polymer comprises one or more polymers selected from the group consisting of: cellulose acetate, dextran, nitrocellulose, ethyl cellulose, carboxymethyl cellulose, cellulose acetate phthalate, cellulose acetate propionate, cellulose acetate butyrate, hydroxypropyl methylcellulose (HPMC), polyethylene glycol (PEG), polyvinyl alcohol, polyvinyl alcohol-polyethylene glycol graft copolymer, silicone dioxide and derivatives thereof.
  • said polymer is cellulose acetate.
  • said polymer is cellulose acetate phthalate.
  • the polymer consists a single polymer.
  • the polymer consists of a mixture of polymers.
  • the plasticizer is selected from the group consisting of: 1 ,2-cyclohexane dicarboxylic acid di-isononyl ester, bis(2-ethylhexyl) sebacate, acetyl tributyl citrate, triethylhexyl trimellitate, triethyl citrate, ethyl lactate, alkylsulfonic acid phenylester, dioctylphthalate, polylactide, ethyl hexyl phthalate, polyvinyl pyrrolidone (povidone) and combinations thereof.
  • the plasticizer is selected from the group consisting of: 1 ,2-cyclohexane dicarboxylic acid di-isononyl ester, bis(2-ethylhexyl) sebacate, acetyl tributyl citrate, triethylhexyl trimellitate, triethyl citrate, ethyl lac
  • said plasticizer is 1 ,2-cyclohexane dicarboxylic acid di-isononyl ester.
  • plasticizer and “binder”, as used herein, are interchangeable.
  • diazotizing agent refers to an agent which forms a diazonium salt in the presence of nitrites.
  • a diazotizing agent includes, but is not limited to, p-arsanilic acid, , methyl anthranilate, sulfanilamide, sulfanilic acid, 2-amino-6-methoxy benzothiazole, 2-amino- benzothiazole, 2-amino-6-nitro benzothiazole, 2-amino-6- ethoxybenzothiazole, 2-amino-6-methylbenzothiazole, 2-amino-3,5-dinitrothiophene, 4-[(4- methyl- 1 -piperazinyl)sulfonyl]aniline, 4-( ⁇ 4'-[(4-aminophenyl)sulfonyl] [1,1 '-biphenyl]-4- yl ⁇ sulfonyl)aniline, Sulfadimethoxine, sulfathiazole, Sodium sulfanilate hydrate or any aromatic primary amine with a sul
  • the diazotizing agent is sulfanilamide.
  • the diazotizing agent is 4-[(4-methyl-l-piperazinyl)sulfonyl]aniline.
  • the diazotizing agent is 4-( ⁇ 4'-[(4-aminophenyl)sulfonyl][l, -biphenyl]-4- yl ⁇ sulfonyl)aniline.
  • a coupling agent refers to an agent which forms an azo dye in the presence of a diazonium salt.
  • a coupling agent includes, but is not limited to, N-(l- Naphthyl)ethylenediamine dihydrochloride, 4-( ⁇ 4'-[(4-aminophenyl)sulfonyl] [1,1 '-biphenyl]- 4-yl ⁇ sulfonyl)aniline, 4-[(4-methyl-l-piperazinyl)sulfonyl]aniline, 1-Naphthylamine, and 2- (1-Naphthylamino) ethanol.
  • the coupling agent is N-(l- Naphthyl)ethylenediamine dihydrochloride.
  • the indicator composition further comprises at least one component selected from the group consisting of: a pH indicator, a dye, an antioxidant, an acid, a trap and a wetting agent.
  • the indicator composition further comprises a pH indicator.
  • the pH indicator is selected from: leucomalachite green and gentian violet.
  • the pH indicator is leucomalachite green.
  • the pH indicator is gentian violet.
  • said pH indicator has a transition pH range between 0-4.0.
  • said pH indicator has a transition pH range between 8.0-14.0.
  • the amount of the pH indicator in the indicator composition is within the range of 0.0005% to 1%.
  • the indicator composition further comprises a dye. The dye may serve as a reactant which changes color upon exposure to nitrites.
  • the dye may further serve to change the indicative color shade of the indicator composition.
  • the dye is selected from the group consisting of: Indigo (2,2'-Bis(2,3-dihydro-3- oxoindolyliden)), Trypan Blue ((3 ⁇ ,3' ⁇ )-3,3'-[(3,3'- dimethylbiphenyl-4,4'-diyl)di( 1 Z)hydrazin-2-yl- 1 -ylidene]bis(5 -amino-4-oxo-3 ,4- dihydronaphthalene-2,7-disulfonic acid), Food Blue No.
  • the amount of the dye in the indicator composition is within the range of 0.05% to 2.5%. According to yet another embodiment, the amount of the dye in the indicator composition is within the range of 0.005% to 2.5%.
  • the indicator composition further comprises an anti-oxidant.
  • the anti-oxidant is selected from the group consisting of titanium dioxide pigments, zinc oxide, polyvinyl alcohol, ascorbic acid, D-Isoascorbic acid, t-butyl hydroquinone, polystyrene and vitamin E.
  • the amount of the anti-oxidant in the indicator composition is within the range of 10% to 20%.
  • the amount of the anti-oxidant in the indicator composition is within the range of 1% to 20%.
  • the amount of the anti-oxidant in the indicator composition is within the range of 0.1% to 20%.
  • the anti-oxidant is polystyrene. According to yet another embodiment, the anti-oxidant is t-butyl hydroquinone. According to yet another embodiment, the anti-oxidant is polyvinyl alcohol.
  • the indicator composition further comprises an acid.
  • the acid provides an acidic environment, which is favorable for the Griess reaction.
  • the acid may also prevent interference from ammonia in urine. Ammonia reacts with nitrites and thus reduces the sensitivity of the indicator composition.
  • the acid is selected from the group consisting of: citric acid, tartaric acid, adipic acid, picric acid, methacrylic acid, stearic acid and acetic acid.
  • the acid is citric acid. Each possibility represents a separate embodiment of the present invention.
  • the acid is a salt derivative of an acid selected from the group consisting of: citric acid, tartaric acid, adipic acid, picric acid and acetic acid.
  • the acid is a salt derivative of citric acid.
  • the amount of the acid in the indicator composition is within the range of 0.1% to 10%.
  • the indicator composition further comprises a trap.
  • the trap is a glucose trap or an ascorbic acid trap.
  • the trap is selected from beta-cyclodextrin and pentasodium pentetate. Each possibility represents a separate embodiment of the present invention.
  • the trap is beta-cyclodextrin.
  • the amount of the trap in the indicator composition is within the range of 0.5% to 2.5%.
  • the amount of the trap in the indicator composition is within the range of 0.05% to 2.5%.
  • the trap is intended to mask interference to the stable color indication.
  • the interference may be caused by glucose and/or ascorbic acid.
  • the trap bind to at least one of glucose and ascorbic acid, thereby masking interference.
  • the indicator composition further comprises a photo-resistant agent.
  • the photo-resistant agent is 4- methacryloxy-2-hydroxy benzo- phenone.
  • the amount of the photo-resistant agent in the indicator composition is within the range of 0.1% to 1%.
  • the photo-resistant agent protects the indicator-composition from damages related to ultra-violet radiation, such as, but not limited to, formation of free radicals.
  • the indicator composition further comprises a wetting agent.
  • the wetting agent may act to increase the hydrophilic characteristics of the absorbent substrate. The increase in hydrophilic characteristics improves the interaction between the hydrophilic urine and the absorbent substrate.
  • the wetting agent is selected from the group consisting of: glycerol, 2-ethoxy ethanol and sodium carboxy methyl starch. Each possibility represents a separate embodiment of the present invention.
  • the wetting agent is glycerol.
  • the amount of the wetting agent in the indicator composition is within the range of 1% to 25%.
  • the amount of the wetting agent in the indicator composition is within the range of 1% to 40%.
  • the sensitivity of the indicator composition to nitrites is high and an indication is obtained even for traces of nitrites in the urine.
  • the sensitivity of said indicator composition to nitrites is in the range of 0.01 to 2 mg/dL.
  • the indicator composition is incorporated in an absorbent substrate.
  • incorporating the indicator composition in an absorbent substrate forms the article of the invention.
  • the indicator composition is applied onto the absorbent substrate by spraying, printing or coating.
  • the absorbent substrate is incorporated in a device selected from the group consisting of: gauze, panty shield, hygienic napkin, diaper, inter-labial absorbent structure, an absorbent sticker and a combination thereof. Each possibility represents a separate embodiment of the present invention.
  • absorbent sticker refers to an absorbent material comprising a sticky surface.
  • the absorbent sticker is configured to stick to any other absorbent substrate known in the art.
  • the indicator composition is initially applied to an absorbent sticker and the sticker is then applied to another absorbent substrate.
  • the absorbent substrate may be of many suitable absorbent materials known in the art such as polyester membranes, polypropylene membranes, cellulose membranes, paper, cotton and linen.
  • the absorbent substrate comprises a fabric material.
  • the fabric material consists of a woven fabric.
  • the fabric material consists of a non-woven fabric.
  • the fabric material consists of a hydrophilic fabric.
  • the fabric material consists of a hydrophobic fabric.
  • the fabric material is selected from the group consisting of: woven fabric, non- woven fabric, hydrophilic fabric material, hydrophobic fabric material and combinations thereof.
  • the diagnostic article further comprises a protective film.
  • the protective film may improve the stability of the indicator composition throughout the indicator composition's shelf-life.
  • the protective film is configured to prevent the indicator composition from providing a color reaction until the protective film is dissolved, preferably, until the protective film dissolves upon contact with urine.
  • the protective film is configured to prevent the indicator composition from reacting with air and/or nitrites until the protective film is dissolved, preferably, until the protective film dissolves upon contact with urine.
  • the protective film is configured to dissolve upon contacting urine.
  • the protective film comprises a polymer selected from the group consisting of: polyvinyl alcohol, polyethylene glycol, hydrous magnesium silicate, hydroxypropyl methyl cellulose, sodium starch glycollate, Polyethylene glycol (PEG), xanthan gum and a combination thereof.
  • the protective film comprises talc.
  • the protective film comprises talc and a polymer selected from the group consisting of: polyvinyl alcohol, polyethylene glycol, hydrous magnesium silicate, hydroxypropyl methyl cellulose, sodium starch glycollate, Polyethylene glycol (PEG), xanthan gum and a combination thereof.
  • talc refers to a hydrated magnesium silicate having a chemical formula selected from the group consisting of: H 2 Mg 3 (Si0 3 )4 and Mg 3 Si 4 Oi 0 (OH) 2 .
  • the protective film has one or more of the following properties: water soluble, biocompatible, partially or completely impermeable to gases or a combination thereof.
  • the protective film is applied onto the absorbent substrate, thereby covering and/or protecting the absorbent substrate.
  • the protective film is applied onto the absorbent substrate by coating, spraying or any suitable method known in the art
  • the protective film is applied onto the absorbent substrate wherein the absorbent substrate already includes the indicator composition.
  • the article of the invention is prepared as follows: first, the indicator composition is applied onto the absorbent substrate, thereby forming an absorbent substrate containing the indicator composition. Second, the absorbent substrate containing the indicator composition is covered, or otherwise protected, with the protective film.
  • the present invention discloses a method for detecting urinary tract infection in a subject.
  • a diagnostic article comprising an absorbent substrate, configured to absorb urine, and an indicator composition is used.
  • the indicator composition comprises a polymer, a plasticizer, a diazotizing reagent and a coupling agent.
  • the indicator composition provides a color reaction in the presence of nitrites in the absorbed urine.
  • the article is mounted in a way allowing receiving urine from the subject and the indicator composition is contacted with urine.
  • the color of the article, or parts thereof which incorporate the diagnostic composition is detected, thereby allowing to determine whether an abnormal level of nitrites, indicative of UTI, is present in the urine.
  • the article used in the method further comprises mounting means for placing the absorbent substrate in a position to receive urine secreted from the subject, such as adhesive strips.
  • the color reaction of the article is monitored by comparing color in the article to a color scale provided by a manufacturer.
  • the method of the present invention may be used by any subject in need thereof, men, women and infants, whether autonomously or with the help of a care provider.
  • the care provider may have limited medical training.
  • the subject may mount the article within undergarments (such as in the case of a panty shield) or using mounting means such as adhesive strips (in the case of a diaper).
  • the article of the present invention is prepared as follows: initially, a polymer solution is pre-formed, by mixing dry polymer and a plasticizer with a solvent. Typically, 1.5 g of polymer is dissolved in between 4 ml and 60 ml of solvent, preferably between 30 ml and 50 ml solvent. Next, the essential components of the indicator composition are added to the solution, namely a diazotizing agent and a coupling agent (the essential components may be separately dissolved in a solvent prior to being added to the polymer solution). Optionally, at least one of the additional components is added into the solution, namely, a pH indicator, a dye, an anti-oxidant, an acid, a trap and a wetting agent.
  • the indicator solution is applied by suitable means to an absorbent substrate.
  • Application may be carried out by any suitable method known in the art, such as, spraying or spreading the mixture onto an absorbent substrate or by dipping the absorbent substrate in the mixture.
  • a protective film is further applied to the absorbent substrate by any of the abovementioned application methods.
  • the solvent may be selected from the group consisting of: water, isopropyl alcohol, ethanol, acetone and a combination thereof.
  • the solvent further comprises a buffer.
  • the buffer maintains a transition pH range of 2.0-5.0.
  • the buffer comprises one or more of the buffers selected from the group consisting of: citrate buffer, phosphate buffer, acetic buffer, tartaric buffer and adipic buffer.
  • the absorbent substrate may be further integrated into another article to form the final article.
  • the absorbent material which contains the indicator composition may be incorporated into a panty liner.
  • Example 1 Indicator compositiosn containing polyvinyil alcohol.
  • Example 2 Indicator compositions containing sulfanilamide as a diazotizing
  • Example 3 Indicator-compositions containing glycerol as a wetting agent.
  • Example 4 Indicator compositions containing 4-[(4-methyl-l- piperazinyl)sulfonyl] aniline as a diazotizing agent.
  • An indicator composition for detection of nitrites in urine containing 4-[(4-methyl-l- piperazinyl)sulfonyl]aniline as a diazotizing agent was prepared in a solution of water and acetone.
  • the components of several indicator composition, including the solvents, are described in Tables 16-20.
  • Example 5 Indicator compositions containing 4-( ⁇ 4'-[(4-aminophenyl)sulfonyl] [l,l'- biphenyl]-4-yl ⁇ sulfonyl)aniline as a diazotizing agent.
  • An indicator composition for detection of nitrites in urine containing 4-( ⁇ 4'-[(4- aminophenyl)sulfonyl][l, -biphenyl]-4-yl ⁇ sulfonyl)aniline as a diazotizing agent was prepared in a solution of water and acetone.
  • the components of several indicator compositions, including the solvents, are described in Tables 21-23.
  • Example 6 Indicator compositions containing 2-amino 6-methoxybenzothiazole as a diazotizing agent.
  • An indicator composition for detection of nitrites in urine containing 2-amino 6- methoxybenzothiazole as a diazotizing agent was prepared in a solution of water and acetone.
  • the components of the indicator composition, including the solvents, are described in Table 24. Table 24:
  • Example 7 Indicator compositiosn containing sulfadimethoxine as a diazotizing agent.
  • An indicator composition for detection of nitrites in urine containing Sulgadimethoxine as a diazotizing agent was prepared in a solution of water and acetone.
  • the components of the indicator composition, including the solvents, are described in Table 25.
  • Example 8 Indicator compositiosn containing Sodium sulfanilate hydrate as a diazotizing agent.
  • An indicator composition for detection of nitrites in urine containing Sodium sulfanilate hydrate as a diazotizing agent was prepared in a solution of water and acetone.
  • the components of the indicator composition, including the solvents, are described in Table 26. Table 26:
  • Example 9 Indicator compositions containing methyl anthranilate.
  • An indicator composition for detection of nitrites in urine containing Methyl anthranilate was prepared in a solution of water and acetone.
  • the components of the indicator composition, including the solvents, are described in Table 27.
  • Example 10 Formulation of indicator-composition containing Beta-cyclodextrin as a trap.
  • Example 11 Detection of nitrites in nitrite solutions using an indicator composition
  • Polyester non-woven fabric was coated with a solution containing an indicator composition of the formulation laid down in Table 31.
  • the fabric was dipped into the indicator composition solution and dried using hot air, resulting in a white-colored fabric.
  • the fabric was cut into pieces, and each piece was placed in a diaper.
  • Each piece was treated with a water-based nitrite solution containing either 0.01 mg/dL, 0.025 mg/dL or 0.05 mg/dL nitrite. A clear purple stain appeared on each piece and remained stable for 24 hours.
  • Similar results were obtained when the fabric was coated with a commercial mixture of OPADRY II - Colorcon (5% in distilled water), as a protective-film, prior to treatment with the nitrite solution.
  • Similar results were obtained when coated fabrics were treated with human urine samples to which nitrites were added (0.01 mg/dL, 0.025 mg/dL or 0.05 mg/dL).

Abstract

The present invention relates to the field of medical diagnostics and more specifically, to an improved identification of urinary tract infection (UTI), using a urine-testing article comprising nitrite-indicator polymer matrix to identify nitrites in urine. The present invention further describes methods for preparing and using said urine-testing article.

Description

ARTICLES AND METHODS FOR MONITORING URINARY TRACT
INFECTION
FIELD OF THE INVENTION
The present invention relates to the field of medical diagnostics and more specifically, to an improved identification of urinary tract infection (UTI), using a urine-testing article comprising nitrite-indicator polymer matrix to identify nitrites in urine. The present invention further describes methods for preparing and using said urine-testing article.
BACKGROUND OF THE INVENTION Urinary tract infections are most common among females, with a high recurrence rate of about 25%-30%. UTI are considered the most frequent bacterial infections in women and it is estimated that about one third of all adult women have experienced an episode of symptomatic cystitis (bladder infection) at least once. UTI are common in infants and among young boys and young girls, albeit with considerably lower estimates. Most episodes of UTI are caused by bacterial infection; however in many cases the etiology is not clear. Typically, diagnosis of UTI includes dipping a dipstick in a urine sample for identifying elements, such as, leukocyte esterase or nitrites, which are indicative of infection. Common diagnosis also includes examining proliferation of microorganisms in a urine culture. The presence of nitrites in urine is a well-known marker for UTI, as the majority of bacteria that cause UTI induce reduction of nitrates to nitrites, thus causing a substantial elevation in nitrite level in the subject's urine.
One of the approaches to detecting nitrites in urine includes the use of a diazotizing agent and a coupling agent. For example, U.S. Patent No. 4,631,255 discloses a composition for assaying for nitrites comprising an acid component, a diazotizing agent and a coupling agent, wherein said composition changes color upon contacting a urine sample containing nitrites. Most diagnostic tools for UTI require the involvement of health care professionals, and therefore are used only in medical facilities. There are, however, diagnostic tools for UTI that are suitable for personal use at home. For example, U.S. Patent No. 6,203,496 discloses a disposable article, such as, a diaper, a panty liner and a pad, for UTI preliminary diagnosis even by personnel having limited medical training. The article comprises one or more chemical reagents that change color upon contact with urine containing abnormal levels of substances, including nitrites. Suitable reagents include naphthyl ester, diazonium salt, arsenilic acid and tetrahydrobenzo (h) quinolin.
U.S. Patent No. 6,464,635 discloses a diaper comprising chemicals for visual examination of urine status, other than UTI, based on the level of various substances such as nitrates, bilirubin, ketones, blood, proteins, urobilinogen and glucose.
In products which are intended for home use, the stability of a color-based indication is of high importance. A secretion-monitoring article for identifying a secreted biological fluid, other than urine, providing a stable visual indication is disclosed in U.S. Patent No. 6,921,647. The article comprises a pH indicator agent, a polymer and an ion-balance reagent where the indication is stable for at least 48 hours.
There is currently a need for a urinary tract infection detection system, which can be easily used by both a health care giver and an untrained user and which would present a clear, long lasting and non-leaching indication.
SUMMARY OF THE INVENTION
The present invention provides indicator compositions, diagnostic articles and uses thereof for detecting the presence of nitrites in a subject's urine, thereby diagnosing urinary tract infections. The invention relies on the unexpected discovery that incorporating a chemically-based detection system for nitrites with a polymer provides a stable color indication. The resulting indication does not leach and remains indicative for at least 24 hours. Moreover, the color indication is maintained even after several cycles of drying and re- wetting of the indicator system. Advantageously, the indication provided by the article of the invention is not affected by interfering substances and conditions, such as glucose, ascorbic acid and changes in urine pH. Such an indicator composition would enable a reliable early detection of urinary tract infection at the comfort of the user's home, would accelerate treatment at early stages of the inflammation and thereby may prevent unnecessary discomfort and pain for the subject. According to one aspect, the present invention provides an indicator composition comprising a polymer, a plasticizer, a diazotizing reagent and a coupling agent, wherein said indicator composition provides a color indication in the presence of nitrites in urine.
According to another aspect, the present invention provides a diagnostic article for detection of urinary tract infection in a subject, comprising an absorbent substrate configured to absorb urine and an indicator composition comprising a polymer, a plasticizer, a diazotizing reagent and a coupling agent, wherein said indicator composition provides a color indication in the presence of nitrites in said absorbed urine.
According to another aspect, the present invention provides a diagnostic article for detection of urinary tract infection in a subject, comprising an absorbent substrate configured to absorb urine and the indicator composition of the invention, wherein said indicator composition provides a color indication in the presence of nitrites in said absorbed urine.
According to one embodiment, the indicator composition further comprises at least one component selected from the group consisting of: a pH indicator, a dye, an anti-oxidant, an acid, a trap, a photo-resistant agent and a wetting agent. It is to be understood that during preparation of the indicator composition, a solvent is added in order to obtain a homogenous solution of all the components in the composition. The solvent may contain a buffer. In some embodiment, the buffer maintains an acidic pH. In other embodiments, the buffer maintains a transition pH range of 2.0-5.0. However, upon applying the indicator composition onto an absorbent substrate (thereby forming the article of the invention), the solvent is evaporated.
According to another embodiment, the amount of the polymer in the indicator composition is within the range of 30% to 90%, the amount of the plasticizer in the indicator composition is within the range of 5% to 50%, the amount of the diazotizing reagent in the indicator composition is within the range of 0.1% to 12%, the amount of the coupling agent in the indicator composition is within the range of 0.01% to 5%, wherein the percentages presenting the amount of each component in the indicator composition refer to weight/weight percentages based on the total weight of the indicator composition and the total weight of the indicator composition equals 100%. According to yet another embodiment, the indicator composition further comprises pH indicator wherein the amount of the pH indicator in the indicator composition is within the range of 0.0005% to 1%.
According to yet another embodiment, the indicator composition further comprises a dye wherein the amount of the dye in the indicator composition is within the range of 0.05% to 2.5%.
According to yet another embodiment, the indicator composition further comprises an anti-oxidant, wherein the amount of the anti-oxidant in the indicator composition is within the range of 1% to 20%.
According to yet another embodiment, the indicator composition further comprises an acid, wherein the amount of the acid in the indicator composition is within the range of 0.1%- 10%.
According to yet another embodiment, the indicator composition further comprises a trap, wherein the amount of the trap in the indicator composition is within the range of 0.5% to 2.5%. According to yet another embodiment, the indicator composition further comprises a wetting-agent, wherein the amount of the wetting agent in the indicator composition is within the range of 1% to 25%.
According to yet another embodiment, the indicator composition further comprises a photo-resistant agent, wherein the amount of the photo-resistant agent in said indicator composition is within the range of 0.1 % to 1 %.
According to yet another embodiment, the sensitivity of the indicator composition to nitrites is in the range of about 0.01 to 2 mg/dL. According to yet another embodiment, said polymer comprises one or more of the polymers selected from the group consisting of: cellulose acetate, dextran, nitrocellulose, ethyl cellulose, carboxymethyl cellulose, cellulose acetate phthalate, cellulose acetate propionate, cellulose acetate butyrate, hydroxypropyl methylcellulose (HPMC), polyethylene glycol (PEG), polyvinyl alcohol, polyvinyl alcohol-polyethylene glycol graft copolymer, silicone dioxide and derivatives thereof.
According to yet another embodiment, said polymer is cellulose acetate.
According to yet another embodiment, said plasticizer is selected from the group consisting of: 1 ,2-cyclohexanedicarboxylic acid di-isononyl ester; bis(2-ethylhexyl) sebacate, acetyl tributyl citrate, triethylhexyl trimellitate, triethyl citrate, ethyl lactate, alkylsulfonic acid phenylester, dioctylphthalate, polylactide, ethyl hexyl phthalate, polyvinyl pyrrolidone (povidone) and combinations thereof.
According to yet another embodiment, said plasticizer is 1 ,2-cyclohexane dicarboxylic acid di-isononyl ester. According to yet another embodiment, said diazotizing agent is selected from the group consisting of: p-arsanilic acid, methyl anthranilate, sulfanilamide, sulfanilic acid, 2- amino-6-methoxy benzothiazole, 2-amino- benzothiazole, 2-amino-6-nitro benzothiazole, 2- amino-6-ethoxybenzothiazole, 2-amino-6-methylbenzothiazole, 2-amino-3,5- dinitrothiophene, 4-[(4-methyl-l-piperazinyl)sulfonyl]aniline, 4-({4'-[(4- aminophenyl)sulfonyl] [1,1 '-biphenyl]-4-yl} sulfonyl)aniline, Sulfadimethoxine, sulfathiazole, Sodium sulfanilate hydrate or any aromatic primary amine with a sulfonyl group and a combination thereof.
According to yet another embodiment, said diazotizing agent is sulfanilamide.
According to yet another embodiment, said diazotizing agent is 4-[(4-methyl-l- piperazinyl)sulfonyl]aniline.
According to yet another embodiment, said diazotizing agent is 4-({4'-[(4- aminophenyl)sulfonyl] [1,1 '-biphenyl]-4-yl} sulfonyl)aniline. According to yet another embodiment, said coupling agent is selected from the group consisting of: N-(1-Naphthyl)ethylenediamine dihydrochloride, 4-({4'-[(4- aminophenyl)sulfonyl] [1,1 '-biphenyl] -4-yl} sulfonyl)aniline, 4- [(4-methyl- 1 - piperazinyl)sulfonyl] aniline, 1-Naphthylamine and 2-(l-Naphthylamino) ethanol.
According to yet another embodiment, the pH indicator has a transition pH range between 0-4.0. According to yet another embodiment, the pH indicator with a transition range between 0-4.0 is selected from: leucomalachite green and gentian violet.
According to yet another embodiment, the pH indicator has a transition pH range between 8.0-14.0.
According to yet another embodiment, said dye is selected from the group consisting of: Indigo (2,2'-Bis(2,3-dihydro-3- oxoindolyliden)), Trypan Blue ((3Ζ,3'Ζ)-3,3'-[(3,3'- dimethylbiphenyl-4,4'-diyl)di( 1 Z)hydrazin-2-yl- 1 -ylidene]bis(5 -amino-4-oxo-3 ,4- dihydronaphthalene-2,7-disulfonic acid), Food Blue No. 1 Aluminum Lake, Alizarin Blue Black B (disodium 4,4'-[(4-hydroxy-9,10-dioxo-9,10-dihydroanthracene-l,3- diyl)diimino]dibenzenesulfonate or other food or cosmetic grade color.
According to yet another embodiment, said dye is Indigo, also known as (2,2'-bis(2,3- dihydro-3- oxoindolyliden).According to yet another embodiment, said anti-oxidant is selected from the group consisting of titanium dioxide pigments, zinc oxide, polyvinyl alcohol, ascorbic acid, D-Isoascorbic acid, t-butyl hydroquinone, polystyrene and vitamin E.
According to yet another embodiment, said trap is selected from the group consisting of: beta-cyclodextrin and pentasodium pentetate.
According to yet another embodiment, said wetting agent is selected from the group consisting of: glycerol, 2-ethoxy ethanol and sodium carboxy methyl starch.
According to yet another embodiment, said wetting agent is glycerol.
According to yet another embodiment, said acid is selected from the group consisting of: citric acid, tartaric acid, adipic acid, picric acid, methacrylic acid, stearic acid and acetic acid. According to yet another embodiment, said acid is a salt derivative of an acid selected from the group consisting of: citric acid, tartaric acid, adipic acid, picric acid and acetic acid.
According to yet another embodiment, said acid is citric acid.
According to yet another embodiment, the photo-resistant agent is 4-methacryloxy-2- hydroxy benzo- phenone.
According to yet another embodiment, said indicator composition provides an indication which is stable, does not leach through several cycles of drying and re-wetting and is indicative for at least 3 hours upon contact with nitrites in urine, preferably for at least 12 hours, preferably for at least 24 hours and preferably for at least 48 hours. According to another embodiment, the indication is stable for at least 3 hours. According to another embodiment, the indication is stable for at least 12 hours. According to another embodiment, the indication is stable for at least 24 hours. According to another embodiment, the indication is stable for at least 48 hours.
According to yet another embodiment, the absorbent substrate is selected from the group consisting of: gauze, panty shield, hygienic napkin, diaper,inter-labial absorbent structure, an absorbent sticker and a combination thereof.
According to yet another embodiment, the absorbent substrate comprises a fabric material. According to yet another embodiment, the fabric material is selected from the group consisting of: woven fabric, non- woven fabric, hydrophilic fabric material, hydrophobic fabric material and combinations thereof.
According to yet another embodiment, the diagnostic article further comprises a protective film separating the indication composition from the environment. According to yet another embodiment, the protective film is configured to dissolve upon contacting urine. According to yet another embodiment, the protective film comprises a polymer selected from the group consisting of: polyvinyl alcohol, polyethylene glycol, hydrous magnesium silicate, hydroxypropyl methyl cellulose, sodium starch glycollate, Polyethylene glycol (PEG), xanthan gum and a combination thereof. According to yet another embodiment, the protective film comprises talc. According to another aspect, the present invention provides a method for detecting urinary tract infection in a subject, the method comprising
(a) providing a diagnostic article comprising an absorbent substrate configured to absorb urine and an indicator composition, wherein said indicator composition comprises a polymer, a plasticizer, a diazotizing reagent and a coupling agent and wherein said indicator composition provides a color indication in the presence of nitrites in said absorbed urine;
(b) mounting said diagnostic article so as to receive urine from the subject;
(c) contacting the indicator composition with urine; and (d) detecting the color indication, thereby determining whether an abnormal level of a nitrites indicative of UTI, is present in the urine.
According to one embodiment detecting comprises observing the color indication of the indicator composition, wherein a color that is different from the color of said indicator composition prior to the step of mounting, is indicative of abnormal level of nitrites. According to another embodiment, contacting the indicator composition with urine takes place upon receiving urine secreted from said subject.
According to yet another embodiment, the diagnostic article comprises a protective film wherein step (c) further comprises removing the protective film.
According to yet another aspect, the present invention provides a method for preparing the article of the invention, the method comprising preparing a solution comprising the indicator composition and a solvent. According to one embodiment, said solvent is selected from the group consisting of: water, isopropyl alcohol, ethanol, acetone and a combination thereof.
Further embodiments, features, advantages and the full scope of applicability of the present invention will become apparent from the detailed description and examples given hereinafter. However, it should be noted that the detailed description, while indicating preferred embodiments of the invention, is given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
DETAILED DESCRIPTION OF THE INVENTION The present invention provides an indicator composition for detecting nitrites, a diagnostic article comprising the indicator composition and a method of using same to detect urinary tract infections in a subject in need thereof. The article and methods of the invention are configured to be used by the user himself or a care taker, and do not require the involvement of personnel with medical training. Furthermore, the article and method of the invention may be applied outside medical facilities, providing theretofore outstanding patient compliance together with stable and reliable indication of UTI. A main advantage of the indicator composition of the invention is the stable indication that it provides. The indication is maintained through cycles of drying and wetting, and is not affected by interfering substances and conditions, such as, glucose, and/or ascorbic acid and/or changes in urine pH. Without being bound to any theory or mechanism, these outstanding qualities are conferred by the specific combination of components in the indicator composition, including the presence of a polymer and a trap among other components.
According to one aspect, the invention provides an indicator composition for detection of urinary tract infection in urine of a subject, the indicator composition comprising a polymer, a plasticizer, a diazotizing reagent and a coupling agent; wherein the indicator composition provides a color reaction in the presence of nitrites in the urine of said subject. According to another embodiment, the color reaction provided by the indicator composition results in s color indication. According to some embodiment, the terms "color reaction" and "color indication" are used interchangeably. According to another aspect, the invention provides a diagnostic article for detection of urinary tract infection in a subject, wherein the diagnostic article comprises an absorbent substrate configured to absorb urine and the indicator composition of the invention. The indicator composition comprises a polymer, a plasticizer, a diazotizing reagent and a coupling agent, wherein the indicator composition provides a color reaction in the presence of nitrites in the absorbed urine. The terms "urinary tract infection" and "UTI" are interchangeably used herein to define an infection of the urinary tract typically induced by bacteria. The bacteria may be Escherichia coli and Staphylococcus saprophyticus among others.
Without being bound to any theory or mechanism, the microorganisms that cause UTI induce reduction of nitrates to nitrites. As a result, the level of nitrites in the infected urine is elevated. Thus, nitrites give an indication of UTI.
According to yet another embodiment, the color indication provided by the indicator composition of the present invention is based on a chemical reaction between nitrites, in the urine, and the indicator composition. Without being bound to any theory or mechanism, the chemical reaction occurs between the nitrites and the diazotizing agent, followed by a coupling reaction, which produces an azo dye. The color of the azo dye is thereby indicative of the presence of nitrites. This type of chemical reaction is based on the Griess reaction which has been described in the art (Jay B. et al., Analytical Chemistry 1979, 51(9): 1493- 1502). Preferably, the color resulting from the reaction with the indicator-composition is stable. According to yet another embodiment, the stable color indication provided by the indicator composition of the invention is distinctive from the color of urine, excrement or blood. Preferably, the color indication is of colors other than brown, red, or yellow. According to yet another embodiment, the color indication is dependent on the combination of diazotizing reagent and coupling agent. According to yet another embodiment, the color of the color indication is selected from the group consisting of: blue, green, orange, pink, purple, violet and white.
Typically, the color of the indication is determined by the diazotizing agent and the coupling agent or their combination. The article of the invention may combine several indications corresponding to different physiological conditions. For example, the article may further include an indication for vaginosis. To this end, the indicator disclosed in U.S. Pat. No. 6,627,394 may be incorporated in the article of the invention. The variety of colors provided by the various diazotizing agents and coupling agents and their combination enable choosing distinct colors for each medical condition diagnosed by the same article. The terms "stable color", "stable color indication" and "stable indication" as used herein, are interchangeable. These terms refer to a color indication which maintains its color, or essentially the same color, over time. According to another embodiment, the stable color indication is a color indication that does not leach through several cycles of drying and re- wetting. According to another embodiment, the stable color indication is a color indication that is indicative for at least 3 hours after contact with nitrites in urine. According to another embodiment, the stable color indication is a color indication that is indicative for at least 12 hours following contact with nitrites in urine. According to another embodiment, the stable color indication is a color indication that is indicative for at least 24 hours following contact with nitrites in urine. According to another embodiment, the stable color indication is a color indication that is indicative for at least 48 hours after contact with nitrites in urine. According to another embodiment, the stable color indication is an indication which remains of essentially the same color, and does not leach or fade through several cycles of drying and re- wetting, for at least 3 hours upon contact with nitrites in urine or a combination thereof. According to yet another embodiment, the polymer of the indicator-composition enables the stable color indication. According to yet another embodiment, the plasticizer changes the plasticity of the polymer.
According to yet another embodiment, the amount of the polymer in the indicator composition is within the range of 30% to 90%, the amount of the plasticizer in the indicator composition is within the range of 5% to 50%, the amount of the diazotizing reagent in the indicator composition is within the range of 0.1% to 12% and the amount of the coupling agent in the indicator composition is within the range of 0.01% to 5%. The percentages presenting the amount of each component in the indicator composition refer to weight/weight percentages based on the total weight of the indicator composition. The total weight of the indicator composition equals 100%. According to yet another embodiment, the amount of plasticizer in the indicator composition is within the range of 15% to 50%. According to yet another embodiment, the density of the plasticizer and wetting agent solutions is within the range of 0.6-1.26 gr/ml. According to yet another embodiment, the density of the plasticizer and wetting agent solutions is within the range of 0.3-1.5 gr/ml. According to another embodiment, the amount of the polymer in the indicator composition is within the range of 30% to 90%, the amount of the plasticizer in the indicator composition is within the range of 5% to 50%, the amount of the diazotizing reagent in the indicator composition is within the range of 0.1% to 28%, the amount of the coupling agent in the indicator composition is within the range of 0.01% to 5%, wherein the percentages presenting the amount of each component in the indicator composition refer to weight/weight percentages based on the total weight of the indicator composition and the total weight of the indicator composition equals 100%.
According to another embodiment, the amount of the polymer in the indicator composition is within the range of 30% to 90%, the amount of the plasticizer in the indicator composition is within the range of 15% to 50%, the amount of the diazotizing reagent in the indicator composition is within the range of 0.1% to 12%, the amount of the coupling agent in the indicator composition is within the range of 0.01% to 5%, wherein the percentages presenting the amount of each component in the indicator composition refer to weight/weight percentages based on the total weight of the indicator composition and the total weight of the indicator composition equals 100%.
According to yet another embodiment, the polymer comprises one or more polymers selected from the group consisting of: cellulose acetate, dextran, nitrocellulose, ethyl cellulose, carboxymethyl cellulose, cellulose acetate phthalate, cellulose acetate propionate, cellulose acetate butyrate, hydroxypropyl methylcellulose (HPMC), polyethylene glycol (PEG), polyvinyl alcohol, polyvinyl alcohol-polyethylene glycol graft copolymer, silicone dioxide and derivatives thereof. According to yet another embodiment, said polymer is cellulose acetate. According to yet another embodiment, said polymer is cellulose acetate phthalate. According to yet another embodiment, the polymer consists a single polymer. According to yet another embodiment, the polymer consists of a mixture of polymers.
According to yet another embodiment, the plasticizer is selected from the group consisting of: 1 ,2-cyclohexane dicarboxylic acid di-isononyl ester, bis(2-ethylhexyl) sebacate, acetyl tributyl citrate, triethylhexyl trimellitate, triethyl citrate, ethyl lactate, alkylsulfonic acid phenylester, dioctylphthalate, polylactide, ethyl hexyl phthalate, polyvinyl pyrrolidone (povidone) and combinations thereof. Each possibility represents a separate embodiment of the present invention. According to yet another embodiment, said plasticizer is 1 ,2-cyclohexane dicarboxylic acid di-isononyl ester. The terms "plasticizer" and "binder", as used herein, are interchangeable. The term "diazotizing agent" as used herein, refers to an agent which forms a diazonium salt in the presence of nitrites. A diazotizing agent includes, but is not limited to, p-arsanilic acid, , methyl anthranilate, sulfanilamide, sulfanilic acid, 2-amino-6-methoxy benzothiazole, 2-amino- benzothiazole, 2-amino-6-nitro benzothiazole, 2-amino-6- ethoxybenzothiazole, 2-amino-6-methylbenzothiazole, 2-amino-3,5-dinitrothiophene, 4-[(4- methyl- 1 -piperazinyl)sulfonyl]aniline, 4-( {4'-[(4-aminophenyl)sulfonyl] [1,1 '-biphenyl]-4- yl}sulfonyl)aniline, Sulfadimethoxine, sulfathiazole, Sodium sulfanilate hydrate or any aromatic primary amine with a sulfonyl group and a combination thereof. Each possibility represents a separate embodiment of the present invention. According to yet another embodiment, the diazotizing agent is sulfanilamide. According to yet another embodiment, the diazotizing agent is 4-[(4-methyl-l-piperazinyl)sulfonyl]aniline. According to yet another embodiment, the diazotizing agent is 4-({4'-[(4-aminophenyl)sulfonyl][l, -biphenyl]-4- yl} sulfonyl)aniline.
The term "coupling agent" as used herein refers to an agent which forms an azo dye in the presence of a diazonium salt. A coupling agent includes, but is not limited to, N-(l- Naphthyl)ethylenediamine dihydrochloride, 4-( {4'-[(4-aminophenyl)sulfonyl] [1,1 '-biphenyl]- 4-yl}sulfonyl)aniline, 4-[(4-methyl-l-piperazinyl)sulfonyl]aniline, 1-Naphthylamine, and 2- (1-Naphthylamino) ethanol. Each possibility represents a separate embodiment of the present invention. According to yet another embodiment, the coupling agent is N-(l- Naphthyl)ethylenediamine dihydrochloride.
According to yet another embodiment, the indicator composition further comprises at least one component selected from the group consisting of: a pH indicator, a dye, an antioxidant, an acid, a trap and a wetting agent.
According to yet another embodiment, the indicator composition further comprises a pH indicator. According to yet another embodiment, the pH indicator is selected from: leucomalachite green and gentian violet. According to yet another embodiment, the pH indicator is leucomalachite green. According to yet another embodiment, the pH indicator is gentian violet. According to yet another embodiment, said pH indicator has a transition pH range between 0-4.0. In yet another embodiment, said pH indicator has a transition pH range between 8.0-14.0. According to yet another embodiment, the amount of the pH indicator in the indicator composition is within the range of 0.0005% to 1%. According to yet another embodiment, the indicator composition further comprises a dye. The dye may serve as a reactant which changes color upon exposure to nitrites. The dye may further serve to change the indicative color shade of the indicator composition. According to yet another embodiment, the dye is selected from the group consisting of: Indigo (2,2'-Bis(2,3-dihydro-3- oxoindolyliden)), Trypan Blue ((3Ζ,3'Ζ)-3,3'-[(3,3'- dimethylbiphenyl-4,4'-diyl)di( 1 Z)hydrazin-2-yl- 1 -ylidene]bis(5 -amino-4-oxo-3 ,4- dihydronaphthalene-2,7-disulfonic acid), Food Blue No. 1 Aluminum Lake, Alizarin Blue Black B (disodium 4,4'-[(4-hydroxy-9,10-dioxo-9,10-dihydroanthracene-l,3- diyl)diimino]dibenzenesulfonate or other food or cosmetic grade color. Each possibility represents a separate embodiment of the present invention. According to yet another embodiment, the amount of the dye in the indicator composition is within the range of 0.05% to 2.5%. According to yet another embodiment, the amount of the dye in the indicator composition is within the range of 0.005% to 2.5%.
According to yet another embodiment, the indicator composition further comprises an anti-oxidant. According to yet another embodiment, the anti-oxidant is selected from the group consisting of titanium dioxide pigments, zinc oxide, polyvinyl alcohol, ascorbic acid, D-Isoascorbic acid, t-butyl hydroquinone, polystyrene and vitamin E. Each possibility represents a separate embodiment of the present invention. According to yet another embodiment, the amount of the anti-oxidant in the indicator composition is within the range of 10% to 20%. According to yet another embodiment, the amount of the anti-oxidant in the indicator composition is within the range of 1% to 20%. According to yet another embodiment, the amount of the anti-oxidant in the indicator composition is within the range of 0.1% to 20%.
According to yet another embodiment, the anti-oxidant is polystyrene. According to yet another embodiment, the anti-oxidant is t-butyl hydroquinone. According to yet another embodiment, the anti-oxidant is polyvinyl alcohol.
According to yet another embodiment, the indicator composition further comprises an acid. The acid provides an acidic environment, which is favorable for the Griess reaction. The acid may also prevent interference from ammonia in urine. Ammonia reacts with nitrites and thus reduces the sensitivity of the indicator composition. According to yet another embodiment, the acid is selected from the group consisting of: citric acid, tartaric acid, adipic acid, picric acid, methacrylic acid, stearic acid and acetic acid. According to yet another embodiment, the acid is citric acid. Each possibility represents a separate embodiment of the present invention.
According to yet another embodiment, the acid is a salt derivative of an acid selected from the group consisting of: citric acid, tartaric acid, adipic acid, picric acid and acetic acid. Each possibility represents a separate embodiment of the present invention. According to yet another embodiment, the acid is a salt derivative of citric acid. According to yet another embodiment, the amount of the acid in the indicator composition is within the range of 0.1% to 10%.
According to yet another embodiment, the indicator composition further comprises a trap. According to yet another embodiment, the trap is a glucose trap or an ascorbic acid trap. According to yet another embodiment, the trap is selected from beta-cyclodextrin and pentasodium pentetate. Each possibility represents a separate embodiment of the present invention. According to yet another embodiment, the trap is beta-cyclodextrin. According to yet another embodiment, the amount of the trap in the indicator composition is within the range of 0.5% to 2.5%. According to yet another embodiment, the amount of the trap in the indicator composition is within the range of 0.05% to 2.5%. The terms "trap" and "scavenger", as used herein, are used interchangeably.
Without being bound by any theory or mechanism, the trap is intended to mask interference to the stable color indication. The interference may be caused by glucose and/or ascorbic acid. According to yet another embodiment, the trap bind to at least one of glucose and ascorbic acid, thereby masking interference. According to yet another embodiment, the indicator composition further comprises a photo-resistant agent. According to yet another embodiment, the photo-resistant agent is 4- methacryloxy-2-hydroxy benzo- phenone. According to yet another embodiment, the amount of the photo-resistant agent in the indicator composition is within the range of 0.1% to 1%. Without wishing to be bound to any theory or mechanism, the photo-resistant agent protects the indicator-composition from damages related to ultra-violet radiation, such as, but not limited to, formation of free radicals.
According to yet another embodiment, the indicator composition further comprises a wetting agent. The wetting agent may act to increase the hydrophilic characteristics of the absorbent substrate. The increase in hydrophilic characteristics improves the interaction between the hydrophilic urine and the absorbent substrate. According to yet another embodiment, the wetting agent is selected from the group consisting of: glycerol, 2-ethoxy ethanol and sodium carboxy methyl starch. Each possibility represents a separate embodiment of the present invention. According to yet another embodiment, the wetting agent is glycerol. According to yet another embodiment, the amount of the wetting agent in the indicator composition is within the range of 1% to 25%. According to yet another embodiment, the amount of the wetting agent in the indicator composition is within the range of 1% to 40%.
The terms "wetting agent" and "surfactant", as used herein, are interchangeable. According to yet another embodiment, the sensitivity of the indicator composition to nitrites is high and an indication is obtained even for traces of nitrites in the urine. According to yet another embodiment, the sensitivity of said indicator composition to nitrites is in the range of 0.01 to 2 mg/dL.
According to yet another embodiment, the indicator composition is incorporated in an absorbent substrate. According to yet another embodiment, incorporating the indicator composition in an absorbent substrate forms the article of the invention. According to yet another embodiment, the indicator composition is applied onto the absorbent substrate by spraying, printing or coating. According to yet another embodiment, the absorbent substrate is incorporated in a device selected from the group consisting of: gauze, panty shield, hygienic napkin, diaper, inter-labial absorbent structure, an absorbent sticker and a combination thereof. Each possibility represents a separate embodiment of the present invention.
The term "absorbent sticker", as used herein, refers to an absorbent material comprising a sticky surface. According to yet another embodiment, the absorbent sticker is configured to stick to any other absorbent substrate known in the art. According to yet another embodiment, the indicator composition is initially applied to an absorbent sticker and the sticker is then applied to another absorbent substrate.
According to yet another embodiment, the absorbent substrate may be of many suitable absorbent materials known in the art such as polyester membranes, polypropylene membranes, cellulose membranes, paper, cotton and linen.
According to some embodiments, the absorbent substrate comprises a fabric material. According to yet another embodiment, the fabric material consists of a woven fabric. According to yet another embodiment, the fabric material consists of a non-woven fabric. According to yet another embodiment, the fabric material consists of a hydrophilic fabric. According to yet another embodiment, the fabric material consists of a hydrophobic fabric. According to yet another embodiment, the fabric material is selected from the group consisting of: woven fabric, non- woven fabric, hydrophilic fabric material, hydrophobic fabric material and combinations thereof.
Details of manufacture of pads, panty shields, sanitary napkins and diapers, including the attachment of indicators to substrates, are well known to those skilled in the art and have been fully described in the prior art, for example, U.S. Pat. Nos. 5,217,444; 5,897,834; 6,149,590 and 8,044,257.
According to yet another embodiment, the diagnostic article further comprises a protective film. Without wishing to be bound to any theory or mechanism, the protective film may improve the stability of the indicator composition throughout the indicator composition's shelf-life. According to yet another embodiment, the protective film is configured to prevent the indicator composition from providing a color reaction until the protective film is dissolved, preferably, until the protective film dissolves upon contact with urine. According to yet another embodiment, the protective film is configured to prevent the indicator composition from reacting with air and/or nitrites until the protective film is dissolved, preferably, until the protective film dissolves upon contact with urine. According to yet another embodiment, the protective film is configured to dissolve upon contacting urine.
According to yet another embodiment, the protective film comprises a polymer selected from the group consisting of: polyvinyl alcohol, polyethylene glycol, hydrous magnesium silicate, hydroxypropyl methyl cellulose, sodium starch glycollate, Polyethylene glycol (PEG), xanthan gum and a combination thereof. Each possibility represents a separate embodiment of the present invention. According to yet another embodiment, the protective film comprises talc. According to yet another embodiment, the protective film comprises talc and a polymer selected from the group consisting of: polyvinyl alcohol, polyethylene glycol, hydrous magnesium silicate, hydroxypropyl methyl cellulose, sodium starch glycollate, Polyethylene glycol (PEG), xanthan gum and a combination thereof. Each possibility represents a separate embodiment of the present invention. As used herein, the term "talc" refers to a hydrated magnesium silicate having a chemical formula selected from the group consisting of: H2Mg3(Si03)4 and Mg3Si4Oi0(OH)2.
According to yet another embodiment, the protective film has one or more of the following properties: water soluble, biocompatible, partially or completely impermeable to gases or a combination thereof.
According to some embodiments, the protective film is applied onto the absorbent substrate, thereby covering and/or protecting the absorbent substrate. According to yet another embodiment, the protective film is applied onto the absorbent substrate by coating, spraying or any suitable method known in the art According to yet another embodiment, the protective film is applied onto the absorbent substrate wherein the absorbent substrate already includes the indicator composition. According to some embodiments, the article of the invention is prepared as follows: first, the indicator composition is applied onto the absorbent substrate, thereby forming an absorbent substrate containing the indicator composition. Second, the absorbent substrate containing the indicator composition is covered, or otherwise protected, with the protective film.
According to another aspect, the present invention discloses a method for detecting urinary tract infection in a subject. According to this aspect, a diagnostic article comprising an absorbent substrate, configured to absorb urine, and an indicator composition is used. The indicator composition comprises a polymer, a plasticizer, a diazotizing reagent and a coupling agent. The indicator composition provides a color reaction in the presence of nitrites in the absorbed urine. According to the method of the present invention, the article is mounted in a way allowing receiving urine from the subject and the indicator composition is contacted with urine. Upon receipt of urine, the color of the article, or parts thereof which incorporate the diagnostic composition, is detected, thereby allowing to determine whether an abnormal level of nitrites, indicative of UTI, is present in the urine.
According to one embodiment, the article used in the method further comprises mounting means for placing the absorbent substrate in a position to receive urine secreted from the subject, such as adhesive strips.
According to another embodiment, the color reaction of the article is monitored by comparing color in the article to a color scale provided by a manufacturer.
The method of the present invention may be used by any subject in need thereof, men, women and infants, whether autonomously or with the help of a care provider. The care provider may have limited medical training. According to yet another embodiment, the subject may mount the article within undergarments (such as in the case of a panty shield) or using mounting means such as adhesive strips (in the case of a diaper).
According to yet another embodiment, the article of the present invention is prepared as follows: initially, a polymer solution is pre-formed, by mixing dry polymer and a plasticizer with a solvent. Typically, 1.5 g of polymer is dissolved in between 4 ml and 60 ml of solvent, preferably between 30 ml and 50 ml solvent. Next, the essential components of the indicator composition are added to the solution, namely a diazotizing agent and a coupling agent (the essential components may be separately dissolved in a solvent prior to being added to the polymer solution). Optionally, at least one of the additional components is added into the solution, namely, a pH indicator, a dye, an anti-oxidant, an acid, a trap and a wetting agent. Once the indicator solution is ready, it is applied by suitable means to an absorbent substrate. Application may be carried out by any suitable method known in the art, such as, spraying or spreading the mixture onto an absorbent substrate or by dipping the absorbent substrate in the mixture. Optionally, a protective film is further applied to the absorbent substrate by any of the abovementioned application methods.
The solvent may be selected from the group consisting of: water, isopropyl alcohol, ethanol, acetone and a combination thereof. According to yet another embodiment, the solvent further comprises a buffer. According to yet another embodiment, the buffer maintains a transition pH range of 2.0-5.0. According to yet another embodiment, the buffer comprises one or more of the buffers selected from the group consisting of: citrate buffer, phosphate buffer, acetic buffer, tartaric buffer and adipic buffer.
Following incorporation of the indicator solution into the absorbent substrate, the solvent is allowed to evaporate. As a result the final article is obtained. Alternatively, the absorbent substrate may be further integrated into another article to form the final article. For example, the absorbent material which contains the indicator composition may be incorporated into a panty liner.
Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.
EXAMPLES
Example 1. Indicator compositiosn containing polyvinyil alcohol.
An indicator composition for detection of nitrites in urine containing polyvynil alcohol was prepared in a solution of water and acetone. The components of several indicator compositions, including the solvents, are described in Tables 1 and 2. Table 1:
Figure imgf000022_0001
Table 2:
Figure imgf000022_0002
Example 2. Indicator compositions containing sulfanilamide as a diazotizing
agent.
An indicator composition for detection of nitrites in urine containing Sulfanilamide as a diazotizing agent was prepared in a solution of water and acetone. The components of several indicator compositions, including the solvents, are described in Tables 3, 4 and 5. Table 3:
Figure imgf000023_0001
Table 4:
Figure imgf000023_0002
Table 5:
Figure imgf000023_0003
Example 3. Indicator-compositions containing glycerol as a wetting agent.
An indicator composition for detection of nitrites in urine containing Glycerol as a wetting agent was prepared in a solution of water and acetone. The components of several indicator compositions, including the solvents, are described in Tables 6-15. Table 6:
Figure imgf000024_0001
Table 7:
Figure imgf000024_0002
Table 8:
Material Function Amount Units
Sulfathiazole Diazotizing agent 0.4 g
N- 1 -napthylethylenediamme dihydrochloride Coupling agent 0.08 g
Water Solvent 8 ml
Acetone Solvent 40 ml
Cellulose acetate phthalate Polymer 1.875 g
Glycerol Wetting agent 0.6 ml
Photo-resistant
4-methacryloxy-2-hydroxy benzo- phenone agent 0.02 g
Polystyrene Anti-oxidant 0.002 g
Dioctylphthalate Plasticizer 50 μΐ Table 9:
Figure imgf000025_0001
Table 10:
Material Function Amount Units
Sulfathiazole Diazotizing agent 0.4 8
N- 1 -napthylethylenediamme dihydrochloride Coupling agent 0.08 8
Water Solvent 8 ml
Acetone Solvent 40 ml
Cellulose acetate phthalate polymer 1.875 8
Glycerol Wetting agent 0.6 ml
Polystyrene Anti-oxidant 0.01 8
Dioctylphthalate Plasticizer 0.2 ml
Table 11:
Figure imgf000026_0001
Table 12:
Material Function Amount Units
Sulfanilamide Diazotizing agent 0.6 8
N- 1 -napthylethylenediamme dihydrochloride Coupling agent 0.06 8
Water Solvent 6 ml
Acetone Solvent 26 ml
Cellulose acetate phthalate Polymer 0.75 8
Glycerol Wetting Agent 0.9 ml
Dioctylphthalate Plasticizer 0.3 ml
Table 13:
Figure imgf000027_0001
Table 14:
Material Function Amount Units
Sulfanilamide Diazotizing agent 0.6 8
N- 1 -napthylethylenediamme dihydrochloride Coupling agent 0.06 8
Water Solvent 6 ml
Acetone Solvent 28 ml
Cellulose acetate phthalate Polymer 0.75 8
Glycerol Wetting agent 0.9 ml
Polystyrene Anti-oxidant 0.02 8
Dioctylphthalate Plasticizer 0.3 ml
Table 15:
Figure imgf000028_0001
Example 4. Indicator compositions containing 4-[(4-methyl-l- piperazinyl)sulfonyl] aniline as a diazotizing agent.
An indicator composition for detection of nitrites in urine containing 4-[(4-methyl-l- piperazinyl)sulfonyl]aniline as a diazotizing agent was prepared in a solution of water and acetone. The components of several indicator composition, including the solvents, are described in Tables 16-20.
Table 16:
Material Function Amount Units
Coupling agent g
4- [(4-methyl- 1 -piperazinyl)sulfonyl] aniline 0.005
Diazotizing agenl g
2-amino-3,5-dinitrothiophene 1% 0.005
Water Solvent 0.5 ml
Acetone Solvent 50.5 ml
Polymer g
Cellulose acetate 1.5
Hexamoll dinch Plasticizer 1 ml
Citric acid Acid 0.025 g Table 17:
Figure imgf000029_0001
Table 18:
Figure imgf000029_0002
Table 19:
Material Function Amount Units
N- 1 -napthylethylenediamme dihydrochloride Coupling agent 0.005 g
Diazotizing agent g
4- [(4-methyl- 1 -piperazinyl)sulfonyl] aniline 0.05
Water Solvent 2 ml
Acetone Solvent 50 ml
Polymer g
Cellulose acetate 1.5
Hexamoll dinch Plasticizer 1 ml Table 20:
Figure imgf000030_0001
Example 5. Indicator compositions containing 4-({4'-[(4-aminophenyl)sulfonyl] [l,l'- biphenyl]-4-yl}sulfonyl)aniline as a diazotizing agent.
An indicator composition for detection of nitrites in urine containing 4-({4'-[(4- aminophenyl)sulfonyl][l, -biphenyl]-4-yl}sulfonyl)aniline as a diazotizing agent was prepared in a solution of water and acetone. The components of several indicator compositions, including the solvents, are described in Tables 21-23.
Table 21:
Material Function Amount Units
Coupling g
N- 1 -napthylethylenediamine dihydrochloride agent 0.01
Diazotizing g
4-( {4'-[(4-aminophenyl)sulfonyl] [1,1 '-biphenyl]-4- agent
yl} sulfonyl)aniline 0.02
Water Solvent 2 ml
Acetone Solvent 52 ml
Polymer g
Cellulose acetate 1.5
Hexamoll dinch Plasticizer 1 ml
Indigo Dye 0.05 g Table 22:
Figure imgf000031_0001
Example 6. Indicator compositions containing 2-amino 6-methoxybenzothiazole as a diazotizing agent.
An indicator composition for detection of nitrites in urine containing 2-amino 6- methoxybenzothiazole as a diazotizing agent was prepared in a solution of water and acetone. The components of the indicator composition, including the solvents, are described in Table 24. Table 24:
Figure imgf000032_0001
Example 7. Indicator compositiosn containing sulfadimethoxine as a diazotizing agent.
An indicator composition for detection of nitrites in urine containing Sulgadimethoxine as a diazotizing agent was prepared in a solution of water and acetone. The components of the indicator composition, including the solvents, are described in Table 25.
Table 25:
Figure imgf000032_0002
Example 8. Indicator compositiosn containing Sodium sulfanilate hydrate as a diazotizing agent.
An indicator composition for detection of nitrites in urine containing Sodium sulfanilate hydrate as a diazotizing agent was prepared in a solution of water and acetone. The components of the indicator composition, including the solvents, are described in Table 26. Table 26:
Figure imgf000033_0001
Example 9. Indicator compositions containing methyl anthranilate.
An indicator composition for detection of nitrites in urine containing Methyl anthranilate was prepared in a solution of water and acetone. The components of the indicator composition, including the solvents, are described in Table 27.
Table 27:
Figure imgf000033_0002
Example 10. Formulation of indicator-composition containing Beta-cyclodextrin as a trap.
An indicator composition for detection of nitrites in urine containing Beta- cyclodextrin as a trap was prepared in a solution of water and acetone. The components of several indicator compositions, including the solvents, are described in Tables 28-30. Table 28:
Figure imgf000034_0001
Table 29:
Figure imgf000034_0002
Table 30:
Figure imgf000034_0003
Example 11. Detection of nitrites in nitrite solutions using an indicator composition
Polyester non-woven fabric was coated with a solution containing an indicator composition of the formulation laid down in Table 31. The fabric was dipped into the indicator composition solution and dried using hot air, resulting in a white-colored fabric. The fabric was cut into pieces, and each piece was placed in a diaper. Each piece was treated with a water-based nitrite solution containing either 0.01 mg/dL, 0.025 mg/dL or 0.05 mg/dL nitrite. A clear purple stain appeared on each piece and remained stable for 24 hours. Similar results were obtained when the fabric was coated with a commercial mixture of OPADRY II - Colorcon (5% in distilled water), as a protective-film, prior to treatment with the nitrite solution. Similar results were obtained when coated fabrics were treated with human urine samples to which nitrites were added (0.01 mg/dL, 0.025 mg/dL or 0.05 mg/dL).
Table 31:
Figure imgf000035_0001
The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without undue experimentation and without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. The means, materials, and steps for carrying out various disclosed functions may take a variety of alternative forms without departing from the invention.

Claims

An indicator composition comprising a polymer, a plasticizer, a diazotizing reagent and a coupling agent, wherein said indicator composition provides a color indication in the presence of nitrites in urine.
The indicator composition of claim 1 , wherein the sensitivity of said indicator composition to nitrites is in the range of 0.01 to 2 mg/dL.
The indicator composition of claim 1 , wherein said indication is stable, for at least 3 hours.
The indicator composition of claim 3, wherein said indication is stable for at least 12 hours.
The indicator composition of claim 3, wherein said indication is stable for at least 24 hours .
The indicator composition of claim 3, wherein said indication is stable for at least 48 hours.
The indicator composition of claim 1 , further comprising at least one component selected from the group consisting of: a pH indicator, a dye, an anti-oxidant, an acid, a trap, a photo-resistant agent and a wetting agent.
The indicator composition of claim 1 , wherein the amount of said polymer in the indicator composition is within the range of 30% to 90%, the amount of said plasticizer in the indicator composition is within the range of 5% to 50%, the amount of said diazotizing reagent in the indicator composition is within the range of 0.1% to 12%, the amount of said coupling agent in the indicator composition is within the range of 0.01% to 5%, wherein the percentages presenting the amount of each component in the indicator composition refer to weight/weight percentages based on the total weight of the indicator composition and the total weight of the indicator composition equals 100%.
9. The indicator composition of claim 7, wherein the amount of the pH indicator in said indicator composition is within the range of 0.0005% to 1%.
10. The indicator composition of claim 7, wherein the amount of the dye in said indicator composition is within the range of 0.05% to 2.5%.
11. The indicator composition of claim 7, wherein the amount of the anti-oxidant in said indicator composition is within the range of 1% to 20%.
12. The indicator composition of claim 7, wherein the amount of the acid in the indicator composition is within the range of 0.1% to 10%.
13. The indicator composition of claim 7, wherein the amount of the trap in said indicator composition is within the range of 0.5% to 2.5%.
14. The indicator composition of claim 7, wherein the amount of the wetting agent in said indicator composition is within the range of 1% to 25%.
15. The indicator composition of claim 7, wherein the amount of the photo-resistant agent in said indicator composition is within the range of 0.1% to 1%.
16. The indicator composition of claim 1, wherein said polymer comprises one or more polymers selected from the group consisting of: cellulose acetate, dextran, nitrocellulose, ethyl cellulose, carboxymethyl cellulose, cellulose acetate phthalate, cellulose acetate propionate, cellulose acetate butyrate, hydroxypropyl methylcellulose, polyethylene glycol, polyvinyl alcohol, polyvinyl alcohol- polyethylene glycol graft copolymer, silicone dioxide and derivatives thereof.
17. The indicator composition of claim 1, wherein said polymer is cellulose acetate.
18. The indicator composition of claim 1, wherein said diazotizing agent is selected from the group consisting of: p-arsanilic acid, methyl anthranilate, sulfanilamide, sulfanilic acid, 2-amino-6-methoxy benzothiazole, 2-amino- benzothiazole, 2-amino-6-nitro benzothiazole, 2-amino-6-ethoxybenzothiazole, 2-amino-6-methylbenzothiazole, 2- amino-3,5-dinitrothiophene, 4-[(4-methyl- 1 -piperazinyl)sulfonyl] aniline, 4-( {4'-[(4- aminophenyl)sulfonyl] [1,1 '-bipheny 1] -4-yl} sulfony l)aniline, sulfadimethoxine, sulfathiazole, sodium sulfanilate hydrate or any aromatic primary amine with a sulfonyl group and a combination thereof.
19. The indicator composition of claim 1, wherein said diazotizing agent is sulfanilamide.
20. The indicator composition of claim 1, wherein said diazotizing agent is 4-[(4-methyl- 1 -piperazinyl)sulfonyl]aniline.
21. The indicator composition of claim 1, wherein said diazotizing agent is 4-({4'-[(4- aminophenyl)sulfonyl] [1,1 '-biphenyl]-4-yl} sulfonyl)aniline.
22. The indicator composition of claim 1, wherein said coupling agent is selected from the group consisting of: N-(1-Naphthyl)ethylenediamine dihydrochloride, 4-({4'-[(4- aminophenyl)sulfonyl] [1,1 '-biphenyl] -4-yl} sulfonyl)aniline, 4- [(4-methyl- 1 - piperazinyl)sulfonyl] aniline, 1-Naphthylamine and 2-(l-Naphthylamino) ethanol.
23. The indicator composition of claim 1, wherein said plasticizer is selected from the group consisting of: 1 ,2-cyclohexane dicarboxylic acid di-isononyl ester, bis(2- ethylhexyl) sebacate, acetyl tributyl citrate, triethylhexyl trimellitate, triethyl citrate, ethyl lactate, alkylsulfonic acid phenylester, dioctylphthalate, polylactide, ethyl hexyl phthalate, polyvinyl pyrrolidone and combinations thereof.
24. The indicator composition of claim 1, wherein said plasticizer is 1 ,2-cyclohexane dicarboxylic acid di-isononyl ester.
25. The indicator composition of claim 7, wherein said pH indicator is selected from: leucomalachite green and gentian violet.
26. The indicator composition of claim 7, wherein said pH indicator has a transition pH range between 0-4.0.
27. The indicator composition of claim 7, wherein said pH indicator has a transition pH range between 8.0-14.0.
28. The indicator composition of claim 7, wherein said dye is selected from the group consisting of: 2,2'-Bis(2,3-dihydro-3- oxoindolyliden), (3Ζ,3'Ζ)-3,3'-[(3,3'- dimethy lbipheny l-4,4'-diyl)di( 1 Z)hydrazin-2-yl- 1 -ylidene]bis(5 -amino-4-oxo-3 ,4 dihydronaphthalene -2,7-disulfonic acid) and disodium 4,4'-[(4-hydroxy-9,10-dioxo- 9, 10-dihydroanthracene- 1 ,3-diyl)diimino]dibenzenesulfonate.
29. The indicator composition of claim 7, wherein said anti-oxidant is selected from the group consisting of: titanium dioxide pigments, zinc oxide, polyvinyl alcohol, ascorbic acid, D-Isoascorbic acid, t-butyl hydroquinone, polystyrene and vitamin E.
30. The indicator composition of claim 7, wherein said trap is selected from the group consisting of: beta-cyclodextrin and pentasodium pentetate.
31. The indicator composition of claim 7, wherein said wetting agent is selected from the group consisting of: glycerol, 2-ethoxy ethanol and sodium carboxy methyl starch.
32. The indicator composition of claim 7, wherein said wetting agent is glycerol.
33. The indicator composition of claim 7, wherein said acid is selected from the group consisting of: citric acid, tartaric acid, adipic acid, picric acid, methacrylic acid, stearic acid and acetic acid.
34. The indicator composition of claim 7, wherein said acid is a salt derivative of an acid selected from the group consisting of: citric acid, tartaric acid, adipic acid, picric acid and acetic acid.
35. The indicator composition of claim 7, wherein said acid is citric acid.
36. The indicator composition of claim 7, wherein said photo-resistant agent is 4- methacryloxy-2-hydroxy benzo- phenone.
37. A diagnostic article for detection of urinary tract infection in a subject, comprising an absorbent substrate configured to absorb urine; and the indicator composition of any one of claims 1-36, wherein said indicator composition provides a color indication in the presence of nitrites in said absorbed urine.
38. The article of claim 37, wherein said absorbent substrate is selected from the group consisting of: gauze, panty shield, hygienic napkin, diaper ,inter-labial absorbent structure, an absorbent sticker and a combination thereof.
39. The article of claim 37, wherein said absorbent substrate comprises a fabric material.
40. The article of claim 39, wherein said fabric material is selected from the group consisting of: woven fabric, non- woven fabric, hydrophilic fabric material, hydrophobic fabric material and combinations thereof.
41. The article of claim 37, wherein the article further comprises a protective film.
42. The article of claim 41, wherein said protective film is configured to dissolve upon contacting urine.
43. The article of claim 41, wherein said protective film comprises a polymer selected from the group consisting of: polyvinyl alcohol, polyethylene glycol, hydrous magnesium silicate, hydroxypropyl methyl cellulose, sodium starch glycollate, polyethylene glycol, xanthan gum and a combination thereof.
44. The article of claim 43, wherein said protective film further comprises talc.
45. A method for detecting urinary tract infection in a subject, the method comprising:
(a) providing a diagnostic article comprising an absorbent substrate configured to absorb urine and an indicator composition, wherein said indicator composition comprises a polymer, a plasticizer, a diazotizing reagent and a coupling agent and wherein said indicator composition provides a color indication in the presence of nitrites in said absorbed urine;
(b) mounting said diagnostic article so as to receive urine from the subject; (c) contacting the indicator composition with urine; and
(d) detecting the color indication, thereby determining whether an abnormal level of a nitrites indicative of urinary tract infection, is present in the urine.
46. The method of claim 45, wherein detecting comprises observing the color indication of the indicator composition, wherein a color that is different from the color of said indicator composition prior to the step of mounting, is indicative of abnormal level of a nitrites.
47. The method of claim 45, wherein the sensitivity of said indicator composition to nitrites is in the range of 0.01 to 2 mg/dL.
48. The method of claim 45, wherein said indication is stable for at least 3 hours.
49. The method of claim 45, wherein said indication is stable for at least 12 hours.
50. The method of claim 45, wherein said indication is stable for at least 24 hours.
51. The method of claim 45, wherein said indication is stable for at least 48 hours.
PCT/IL2012/050209 2011-06-20 2012-06-18 Articles and methods for monitoring urinary tract infection WO2012176202A1 (en)

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