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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
  • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
  • C12Q1/06—Quantitative determination
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
  • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
  • C12Q1/42—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving phosphatase
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2337/00—N-linked chromogens for determinations of peptidases and proteinases
  • C12Q2337/10—Anilides
  • C12Q2337/12—Para-Nitroanilides p-NA
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2337/00—N-linked chromogens for determinations of peptidases and proteinases
  • C12Q2337/30—Naphthyl amides, e.g. beta-NA, 2-NA, 4-methoxy-beta-naphthylamine, i.e. 4MNA

Definitions

• This invention relates generally to systems for determining the presence of oral diseases in mammals, and more particularly, to a colori ⁇ metric test which is useful in determining periodontal disease activity in a human being, or in an animal.
• Periodontal disease is the major affliction of the human dentition. Today, more teeth are lost to the effects of periodontal disease than to caries (tooth decay). Periodontal disease is a group of conditions affecting the gingiva (gum) and the bones that support the teeth. The primary cause of periodontal disease is bacterial plaque which causes an inflammation of the gum which may result in actual destruction of tissue. In some cases, destruction of the bone occurs to the point where teeth lose their attach- ment thereto.
• Periodontal disease there is usually a large accumulation of bacteria in plaque attached to the tooth, both above (supragingival) and below (subgingival) the gum line.
• This plaque can become calcified in its depths, forming what is known as calculus.
• This plaque and associated calculus can create a pocket between the teeth and the gingiva which is characteristic of the disease.
• Periodontal disease is diagnosed by clinical observation of indicators such as the presence and depth of pockets, loss of attachment of the teeth to the bone, and papillary bleeding of the gums. Clinical observations, however, are not always reliable indicators. For example, deep pockets are not necessarily infected by bacteria capable of causing inflammatory tissue destruction (periodonto- pathic bacteria).
• periodontal disease has traditionally been defined as an inflammation of the gums, which means that host tissue is responding to bacteria and/or the products of bacteria, periodontal disease has not been treated like a bacterial infection in the medical sense.
• periodontal disease has not been treated in the art with antimicrobial drugs because the growth of plaque on the teeth appears to be inevitable and since it is external to the body, would not seem to be treatable by systemically administered drugs.
• periodontal disease was specific to one, or several, particularly damaging bacteria. In fact, about 200 species of microbes have been isolated from various plaque samples. Thus, mechanical treatment which requires instrumenting of the tooth to remove accumulated bacterial deposits non- specifically was deemed to be the appropriate means of treating periodontal disease.
• periodontal disease is characterized by a progressive loss of tooth supporting tissue which occurs when the periodontal pocket is colonized by a preponderance of gram negative anaerobic bacteria (see, e.g., Loesche, et al., "Role of Spirochetes in Periodontal Disease,” Host-Parasite Interaction in Periodontal Disease. Genco and Mergengagen, eds., American Society of Microbiology, Washington, D.C., 1982, pages 62-75 and Slots, "Importance of Black Pigmented Bacteroides in Human Periodontal Disease,” Ibid., pages 27-45).
• Spirochetes and black pigmented bacteroides are particularly prominent when pockets bleed upon probing and when there is clinical evidence of disease progression. Thus, the possibility of drug treatment directed towards these anaerobic organisms is raised. In fact, beneficial results have been observed with the use of metronidazole, an antimicrobial effective against anaerobes.
• metronidazole is available underthe trademark FLAGYL from G. D. Searle & Co., Chicago, IL 60680 as well as in generic form from Zenith Laboratories, Inc., Ramsey, NJ.
• the presence of spirochetes can be determined by microscopic examination using either phase contrast or dark field condensers.
• One prior art technique used for determining the presence of periodontal disease involves the microscopic examination of the plaque for determination of the presence of motile forms, mostly spirochetes, and thereby assesses the need for escalating or terminating therapy. See, Keyes, et al., "Diagnosis of Creviculoradicular Infections," Ibid., pages 395-403 and Listgarten, et al., J. Clin. Periodontal.. Vol. 8, pages 122-138 (1981 ). However, no similar microscopic procedure exists for the identification of BPB.
• a simple and inexpensive test for enabling determination of the presence of periodontal disease measures the proteolytic activity of a specimen of suspected periodontopathogenic bacteria is measured. More specifically, this assay method operates to detect the presence of trypsin- like activity, illustratively in a specimen of subgingival plaque.
• a chromogenic test substance comprises an amino acid or a peptide substrate combined with a chromophore which is hydrolyzable by trypsin-like enzymes in the specimen to release a chromophore. Detection of a color change due to said release indicates whether such trypsin-like activity is present, and hence, whether or not microorganisms which may cause periodontal disease are present.
• One problem with the aforementioned technique is that a minimum number of microorganisms should be present in the specimen in order for the results of the test to be a reliable indication of the presence of periodontopathic organisms.
• the test procedure may produce a false negative result even though periodontal disease-producing microorganisms are present in the specimen. Accordingly, it is an object of this invention to provide an improved test for determining the presence of periodontal disease.
• this invention provides an improved test which measures proteolytic activity of periodontopathogenic bacteria.
• a trypsin-like activity is measured since periodonto ⁇ pathogenic organisms, such as T. denticola, B. gingivalis, B. forsythus, and C. gingivalis, are characterized by such activity.
• the proteolytic enzyme produced by the suspected periodontopathic organisms is not trypsin because it is not inhibited by trypsin inhibitors, it does not require calcium, it is not inhibited by EDTA, and it is active at acidic pHs. However, it does react with a peptide substrate (e.g., BANA) that is commonly used to measure trypsin, and hence, the enzyme has been termed as "trypsin-like.”
• a peptide substrate e.g., BANA
• the specimen is additionally assayed for a nonspecific enzymatic marker for indicating whether the specimen contains a predetermined minimum number of microorganisms sufficient to yield a reliable test result.
• a nonspecific enzymatic marker for indicating whether the specimen contains a predetermined minimum number of microorganisms sufficient to yield a reliable test result.
• Analysis of the results of extensive testing has revealed that a population of approximately between 1 and 10 million bacteria in the specimen is sufficient to yield reliable results with the proteolytic enzyme test.
• the test procedure may yield a negative result solely because there were too few bacteria in the specimen.
• This invention provides a second chromophore-containing chromogenic test substance which is enzymatically degradable by the nonspecific marker enzyme to release a chromophore thereby producing an observable color change when at least a predetermined number of microorganisms are present in the sample.
• the nonspecific enzymatic marker is a phosphatase which is present in most, if not all, bacteria found in the flora of the oral cavity.
• the phosphatase enzyme reacts with a second chromogenic test substance to give a positive result, then there are enough organisms in the sample to give a true, reliable test result for the trypsin-like enzyme test.
• the phospha ⁇ tase test is negative, a negative trypsin-like enzyme test would be expected. The clinician is thereby immediately alerted that the sample is too small and that he or she should resample the patient.
• second chromogenic test substances which are enzymatically degradable by phosphatase enzymes include para- and ortho-nitrophenol phosphates, which give good results with alkaline phosphatase, and o-carboxyphenol phosphate which gives good results with acid phosphatase.
• the nonspecific enzymatic marker is a peptidase, and more specifically an aminopeptidase.
• An exemplary second chromogenic test substance which is enzymatically degradable by an aminopeptidase is L-proiine- ⁇ -naphthylamide.
• L-proiine- ⁇ -naphthylamide which is hydrolyzable by an aminopeptidase, can advantageously be used as the second test substance.
• a color developer is added to produce the color change responsive to enzymatic activity.
• a colorimetric assay kit for providing an indication of the presence of periodontal disease.
• the kit is provided with: (I) a first chromogenic test substance specific to a proteolytic enzyme produced by suspected periodontopathogenic bacteria in bacterial flora from the oral cavity of a mammal; and (2) a second chromogenic test substance nonspecific to enzymes produced by the bacterial flora, the second chromogenic test substance being subject to enzymatic degradation in response to a quantity of microorganisms.
• the first chromogenic test substance is specific to trypsin-like enzymatic activity, and is selected from the group consisting of BANA, BAPNA, and 2-arginine-7-amino-4-trifluoromethyl coumarin.
• the second chromogenic test substance is specific to the enzyme phosphatase, and is selected from the group consisting of p-nitrophenol phosphate, o-nitro- phenol phosphate, and o-carboxyl phosphate.
• the second chromogenic test substance is specific to the enzyme peptidase, and is selected from the group consisting of L-proline- ⁇ -naphthylamide, L- leucine- ⁇ -naphthylamide, and L-phenylalanine- ⁇ -naphthylamide.
• the colorimetric assay kit of the present invention is provided, in certain embodiments, with a color developer.
• the color developer is selected from the group consisting of fast garnet, fast blue, and an acidified solution of p-dimethylaminocinnamaldehyde.
• An oral specimen suspected of containing elevated levels of the organisms characteristic of periodontal disease is subjected to a chromo- phore-containing substrate specific to an enzyme produced by the suspected periodontal disease-producing organisms.
• T. denticola, B. gingivalis, B. forsythus, and the spirochetes produce a proteolytic enzyme and/or a peptidase which is capable of hydrolyzing the substrate, thereby releasing a chromophore as a reaction product.
• This released chromophore can then be colorimetrically observed.
• the addition of another chromogenic agent, or color developer is required.
• the presence or absence of a color in response to enzymatic conditions is correlated to elevated bacterial conditions related to active periodontal disease.
• bacterial species known to be associated with periodontal disease the following species: T. denticola, a spirochete; B. gingivalis, the most virulent of the BPB; B. forsythus, an organism often isolated from sites of progressive destruction; and C. gingivalis, an organism associated with periodontal disease in diabetics; all possess a trypsin-like enzyme that can be measured by the hydrolysis of the trypsin substrate N-benzoyl-DL- arginine-2-naphthylamide (BANA). See, Loesche, J ⁇ Oral Microbiol.
• the ability of the plaque to hydrolyze BANA can reflect the presence and/or proportional increase of one or more of these bacterial species in the plaque and therefore provides a measurement of an anaerobic infection in the periodontal plaque.
• the peptide substrate BANA is colorless. Upon incubation with bacteria in the specimen, the chromo ⁇ phore, ⁇ -naphthylamide, is released from its linkage with the carboxyl group of arginine.
• the peptide substrate is benzoyl-DL-arginine-p-nitroanilide (BAPNA) which forms color as it hydrolyses, thereby obviating the need for an additional color developer to demonstrate the enzymatic activity of the specimen.
• BAPNA benzoyl-DL-arginine-p-nitroanilide
• a second chromogenic test substance is provided to demonstrate that the quantity of microorganisms in the specimen exceeds a predetermined number and is, therefore, sufficient to yield reliable results with the proteolytic enzyme test.
• the second chromogenic substrate is hydrolyzable by an enzyme which is present in most, if not all, bacteria found in the flora of the oral cavity so that it can be used as a measure of the quantity of microorganisms present in the specimen.
• enzymatic degradation of the second chromophore- containing substrate produces an observable color change if there are greater than about 1,000,000 microorganisms in the sample, or such other predetermined number as is necessary to give a true, reliable result with the proteolytic and/or peptidase enzyme test.
• the minimum predetermined number depends on the sensitivity of the chromogenic text substance to enzymatic degradation.
• Phosphatase is an enzyme which is prevalent in the flora of the oral cavity. Both alkaline and acid phosphatase, depending upon the pH of the test solution, can be used as nonspecific markers of enzymatic action.
• a phosphatase is an enzyme that catalyzes the hydrolysis and synthesis of phosphoric acid esters and the transfer of phosphate groups from phosphoric acid to other compounds. Therefore, phosphate group- containing substrates such as o-nitrophenol phosphate which can be hydrolyzed by the action of phosphatase to release the chromophore nitrophenol are suitable as the second chromogenic test substances.
• Other illustrative examples are p-nitrophenol phosphate and o-carboxyphenol phosphate.
• the activity of other enzymes commonly found in the bacterial flora of the oral cavity can be used in lieu of the phosphatase activity.
• Illustrative examples are the amino peptidases for proline, leucine, and phenylalanine.
• the second chromogenic substance chosen to be specific to the peptidase must not release a chromophore which will produce a color which will interfere with the ability of the clinician to observe the color change indicative of the trypsin-like activity of suspected periodontopathogenic bacteria.
• samples of plaque are removed with a sterile curette and suspended in an aqueous solution by vigorous agitation in a small, stoppered presterilized vial.
• a stock solution of BANA is prepared by dissolving 44 mg BANA (Sigma Chemical Company, St. Louis, MO) in 1 ml dimethyl sulfoxide (DMSO). Prior to use, the BANA stock solution is diluted with a 1 :100 by volume mixture of O.1 M Tris (hydroxymethyl) aminomethane hydrochloride buffer at pH 8.5. The buffered BANA solution was added to the plaque sample and allowed to incubate overnight. The addition of a drop of fast garnet produced a color, within about 5 minutes.
• pH of the BANA-sample solution can range from between about 5.0 and 8.5.
• Other buffers such as Sorenson phosphate buffer or a phosphate buffer with EDTA, can be used.
• the results show that a nonbuffered solution in pure distilled water works as well as a buffered solution.
• Incubation generally requires a period of time ranging from about less than an hour ⁇ i.e., on the order of about 1 to 5 minutes) to about 24 hours.
• the incubation temperature should be in the range of about 25 °C to 60°C, and preferably approximate ⁇ ly between 37 °C and 55 °C.
• a second chromogenic test substance p-nitrophenol phosphate (pNPP)
• pNPP p-nitrophenol phosphate
• BANA can be added after the pNPP reagent.
• the specimen is then added to the solution containing the reagents and the solution is incubated at about 37°C. Both reagents are initially colorless. However, the action of alkaline phosphatase from microorganisms in the specimen will liberate the chromophore p-nitrophenol. If a sufficient quantity of microorganisms are present in the sample, the solution will observably turn yellow.
• a negative ONP reaction would tell the clinician that the plaque sample is too small to permit reliable results. The clinician would then either retake a sample from the patient, or depending upon the clinical appearance of the gingival tissue, not resample until the next visit. On the other hand, a positive ONP reaction indicates to the clinician that there is sufficient plaque in the sample to proceed with the addition of the fast garnet reagent. If there is no color change after the addition of the fast garnet, the clinician has determined a nondiseased plaque relative to the presence of anaerobic organisms.
• the trypsin-like enzyme substrate comprises BAPNA and the second chromogenic test substance is N-L- proline- ⁇ -naphthylamide (PNA), the naphthylamide derivative for L-proline amino peptidase.
• PNA N-L- proline- ⁇ -naphthylamide
• the BAPNA substrate is prepared as described for BANA and PNA was added thereto. The specimen is incubated with this mixture overnight. Development of a yellow color indicates an anaerobic infection. Failure of a color to develop is an equivocal result in that it indicates either that the specimen was too small or that there is no anaerobic infection. The addition of a color developer, such as fast garnet, solves the dilemma. The development of an orange to red color from the hydrolysis of PNA indicates that enough plaque was present in the sample. If a yellow color develops, then the plaque sample was too small. This is illustrated in the following diagram:
• DIAGNOSIS anaerobic plaque infection - treat Add Fast Garnet patient and/or site
• chromogenic test substances can be substituted for those described herein, as long as the color developed by the nonspecific enzyme reaction does not interfere with the color developed as a result of the specific trypsin-like enzyme reaction.
• An additional specific example is o-carboxyphenol phosphate which is a substrate for acid phosphatase and the ⁇ -naphthylamide derivatives of L-leucine and L-phenylalanine.
• gingivalis strains are active in vitro against L-pyrrolidonyl- ⁇ -naphthylamide, so that this non-peptide chromophore may be of value in the determining the presence of anaerobic periodontal infections.
• All peptide chromophores, mentioned herein, can be purchased from chemical supply houses such as Sigma Chemicals, St. Louis, MO, U.S.A.
• any developers which will demonstrate colorimetrically the release of a chromo- phore can be utilized in the practice of the invention.
• Illustrative examples include, fast gamet-gbc salt (Sigma Chemicals, St. Louis, MO, U.S.A.) chemically designated o-amino-azotoluene-diazoniumsalt; fast blue; and an acidified solution of p-dimethyl-aminocinnamaldehyde.
• proteolytic activity can be measured with a fluorogenic test substance such as 2-arginine-7-amino-4-trifluoro- methyl coumarin derivatives. Proteolytic activity can therefore be demonstrated by the release of a fluorophore, as fluorescence which can be detected with a UV light source. Therefore, the term "chromophore" as used herein should be interpreted broadly to include a substance which absorbs visual or ultraviolet light or which fluoresces. The combination of a peptide substrate with a fluorophore will result in the production of a color change which is observable under UV light when the fluorophore is released. Observable will be the Stokes shift from fluorescent blue to green, for example.
• a fluorogenic test substance such as 2-arginine-7-amino-4-trifluoro- methyl coumarin derivatives.
• the sampling technique included the removal of subgingi- val plaque.
• the sampling techniques contemplated as being effective for the purposes of this invention can be any such method as is known in the art and can be applied to sample any tissue, fluid, or other specimen suspected of being diseased, and therefore includes, by way of example, subgingival plaque, gingival crevicular fluid, supragingival plaque, oral tissue, saliva or oral rinse expectorant, etc. Saliva or oral rinse expectorants, of course, could be concentrated prior to use by any known means.
• the suspected periodontally-diseased specimen would be removed from either the most periodontally involved site per quadrant or from the mesial buccal approxi- mal site of each first molar on patients without any obvious periodontal disease.
• the supragingival plaque at the sample site would be removed and discarded.
• Filter paper, or the like can be placed in the orifice of a gingival crevice to collect gingival crevicular fluid by capillary action. The protein is then eluted off of the filter paper and subjected to the chromogenic test substance.
• a substantial ⁇ ly rigid carrier is provided with at least one porous surface portion to absorb or receive an oral specimen and/or chromogenic test substance, and in some embodiments, a color developer.
• the porous material may be an absorbent fibrous, woven or nonwoven material, such as filter paper.
• the advantages and benefits associated with the use of the test for periodontal disease according to the present invention are considered to be numerous and commercially significant.
• the methods are useful in periodontal therapy as well as initial determination of the presence or absence of an anaerobic infection. It is believed that the present invention is particularly useful in identifying common periodontitis, namely chronic destructive periodontitis, wherein the patient displays significant increases in spirochetes, T. denticola, B. gingivalis, B. forsythus, and C. gingivalis.
• the test of the present invention is also helpful for quantitative evaluation of various stages of treatment of the disease and can aid in a determination of whether treatment has been adequate and whether additional modalities of treatment are warranted.
• the method is particularly suited for use at periodic maintenance visits to determine whether retreatment is necessary. It should further be appreciated that for purposes of this invention, the present test is not limited to human periodontal disease, but can be equally applied to the determination of whether the disease-causing microorganisms are present in mammals in general. As such, the methods of the present invention will find application in the veterinarian sciences.

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