An in vitro diagnostic method

Abstract: The present invention relates to diagnostic methods for detecting or evaluating the presence of periodontal diseases, especially gingivitis, in humans or lower animals by measuring the presence of leukocyte esterase. These diagnostic methods comprise measuring the amount of leukocyte esterase present in the oral cavity of the human or lower animal being diagnosed. The present invention further relates to diagnostic products useful in vivo for detecting or evaluating the presence of periodontal diseases, especially gingivitis, in humans. These diagnostic products contain at least one agent useful in detecting the presence of leukocyte esterase, and a carrier material. These diagnostic products must be sterile and safe for in vivo contact with the tissue in the oral cavity of the human being diagnosed. Finally, the present invention further relates to diagnostic kits useful for detecting or evaluating the presence of periodontal diseases, especially gingivitis, in humans. These kits comprise separate means for collecting a sample of fluid at one or more oral tissue sites in the human being diagnosed, and means for measuring the amount of leukocyte esterase present in the sample collected.

Description: The present invention relates to in vitro diagnostic methods for detecting or evaluating the presence of periodontal diseases (especially gingivitis) in humans or lower animals by measuring the presence of leukocyte esterase. Periodontal diseases, such as, for example, periodontitis, gingivitis, stomatitis, and the like, are inflammatory conditions of the mouth characterized by inflammatory oral tissue changes usually due to local irritation. The destructive inflammatory process involves the interaction between bacteria, food debris, oral leukocytes, and the epithelial attachment around the tooth and periodontal membranes. If allowed to progress unchecked, such inflammatory processes can lead to resorption of the supportive bone around the roots of the teeth and eventual loss of teeth. The early detection of such inflammatory conditions is very important to proper dental treatment. It has been known for many years that at local sites of inflammation the body produces an increased number of leukocytes. References have suggested measuring the presence of leukocytes for evaluating periodontal health, for example: Rams et al., "A Rationale for Management of Periodontal Diseases: Rapid Identification of Microbial 'Therapeutic Targets' with Phase-Contrasts Microscopy", JADA , 106 , pages 803-812 (1983); and Rams et al., "A Rationale for the Management of Periodontal Diseases: Effects of Tetracycline on Subgingival Bacteria", JADA , 107 , pages 37-41 (1983). In addition, diagnostic methods for detecting the presence of the inflammatory periodontal disease condition have been developed based on detecting peroxidase enzymes from polymorphonuclear (PMN) leukocytes as described, for example, in "Diagnostic Method for Detecting Periodontal Disease", EP-A-158796, by Richardson-Vicks Inc., published October 23, 1985. Reagents for assessing periodontal diseases by detecting the presence of peroxidases and/or salivary occult blood in the oral cavity are known, having been described in: Tenovuo and Anttonen, "Application of a Dehydrated Test Strip, HEMASTIX (RTM) , for the Assessment of Gingivitis", J. of Clinical Periodontology , 5 , pages 206-212 (1978); Anttonen et al., "Experimental Gingivitis in Man Determined by Various indices with Special Reference to HEMASTIX (RTM) -Strip Index", Proc. Finn. Dent. Soc. , 74 , pages 41-45 (1978); Ueda et al., "Assessment of Gingival Bleeding with a Salivary Occult Bleeding Test Strip", Bull. Josai Dent. Univ. , 13 (3), pages 628-633 (1984); Yamaoka et al., "Relation between Changes in the Clinical Symptoms in the Value of Salivary Occult Blood Tests in Periodontally Involved Patients - A Study Using a Test - Paper for Saliva Examination (Salivaster Bld)", Nippon Shishubyo Gakkai Kaishi , 27 , (4), pages 912-922 (1985); and Smith et al., "Changes in Salivary Peroxidase Activity Observed During Experimentally-Induced Gingivitis", J. Clin. Periodontol. (Denmark) , 11 , (6), pages 373-378 (1984). Chemical Abstract, Vol. 69 (1968): 104511h discloses detection of nonspecific esterase in gingival tissue in cases of gingivitis and periodontitis. Thus, while there has been much research into methods for detecting periodontal diseases, none of these references disclose or discuss measuring leukocyte esterase as a method for detecting periodontal diseases. In fact, one publication relating to the use of reagent strips to indicate the presence of leukocyte esterase in urine for detecting whether a patient has pyuria (Kusumi et al., JAMA , 245 (16), pages 1653-1655 (1981) specifically speculates that the leukocyte esterase test "cannot, however, be used to test sputum, since saliva is rich in esterase activity." Therefore, the present invention is clearly surprising in light of the published literature. An object of the present invention is to provide in vitro methods for detecting the presence of periodontal diseases (especially gingivitis) in humans or lower animals. Another object is to provide methods for evaluating the severity of periodontal diseases (especially gingivitis) in humans or lower animals. An object of the present invention Is also to provide in vitro methods for following diseased gingival sites for improvement during the course of therapy; and for targeting gingival sites that are susceptible to gingivitis, prior to overt inflammation, for further monitoring or preventative treatment. These and other objects of the present invention will become readily apparent from the detailed description which follows. All percentages and ratios used herein are by weight unless otherwise specified. According to the present invention there is provided an in vitro diagnostic method for detecting or evaluating the presence of periodontal diseases in humans or lower animals, wherein said method comprises collecting a sample of saliva from the oral carity or a sample from the gingival site to be diagnosed and measuring the mount of leukocyte esterase present in the sample utilizing an indoxyl carbonic acid ester which is hydrolyzable by leukocyte esterase to form indoxyl. The present invention relates to diagnostic methods for detecting or evaluating the presence of periodontal diseases (especially gingivitis) in humans or lower animals. "Detecting or evaluating the presence of periodontal diseases", as used herein, means detecting the presence of, or evaluating the severity of, periodontal diseases. The term "periodontal diseases", as used herein, means inflammatory conditions of the mouth, whether or not discernible to the naked eye, including such inflammatory conditions as periodontitis, stomatitis, and gingivitis. The method of the present invention is particularly useful for detecting the presence of gingivitis which is not readily discernible to the naked eye, and for evaluating the severity of gingivitis whether or not discernible to the naked eye. Thus, the methods of the present invention are especially suited for predicting the onset of gingivitis and/or for quantitatively or semi-quantitatively indicating the degree of the severity of gingivitis. As a result of this early detection, early dental treatment can be initiated to ensure rapid return to proper dental health. "Measuring the amount of leukocyte esterase present in the sample of saliva from the oral cavity or the sample from the gingival site to be diagnosed' as used herein, means either simply detecting the presence of leukocyte esterase; or quantitatively or semi-quantitatively measuring the concentration of leukocyte esterase in the sample taken from the oral cavity. Products and/or reagents utilized for measuring the amount of leukocyte esterase present in the oral cavity are described more fully hereinafter. The methods of the present invention are carried out by either (1) in vitro measurement of a saliva sample; or (2) in vitro measurement of a sample from a specific gingival site or sites. Each of these procedures is further elaborated immediately hereinafter to illuminate and exemplify the methods of the present invention. In vitro measurement of the leukocyte esterase activity in saliva samples is accomplished by utilizing commercially-available leukocyte esterase reagent strips suitable for in vitro evaluation of leukocyte esterase activity. The saliva sample may be expectorated directly onto the strip, or, preferably, is diluted in water to a certain volume and tested by dipping the reagent strip in the dilute saliva sample. It is further preferred that the subject to be diagnosed does not eat within one hour prior to the sample collection. Examples of commercially-available leukocyte esterase reagent strips are CHEMSTRIP (RTM) 9 and CHEMSTRIP (RTM) LN (both sold by Bio-Dynamics, Indianapolis, Indiana); and MULTISTIX (RTM) 2 Reagent Strips and AMES LEUKOSTIX (RTM) (both available from Ames, Division of Miles Laboratory, Elkhart, Indiana). Techniques for using these commercially-available leukocyte esterase reagent strips are well known from their use for in vitro urine analysis (e.g., Scheer, "The Detection of Leukocyte Esterase Activity in Urine with a New Reagent Strip", Am. J. Clin. Pathol. , 87 (1), pages 86-93 (1987)). These known urine analysis techniques are applicable to the in vitro methods of the present invention. All of these commercially-available leukocyte esterase reagent strips contain an indoxyl carbonic acid ester which is hydrolyzed to indoxyl by leukocyte esterase. The indoxyl thus formed reacts with a diazonium compound in the strip to produce a color which indicates the presence of the leukocyte esterase. The degree of darkening of the strips is a semi-quantitative indication of the amount of leukocyte esterase present in the saliva sample, and hence for the methods of the present invention is an indication of the severity of the periodontal disease. The presence of color change when no periodontal diseases are readily evident to the naked eye may indicate the onset of gingivitis. In vitro measurement of a sample obtained from a specific gingival site is a preferred method of the present invention. The sample collection may be accomplished in many ways, but preferred is flushing a region with fluid and then micropipetting a sample of fluid directly from the site of the gingival surface, or more preferably swabbing the gingival site. It is also preferred for these in vitro measurements that the subject to be diagnosed rinse his mouth or brush his teeth within one hour prior to sample collection. The sample collected may be applied directly to a commercially-available leukocyte esterase reagent strip such as described hereinbefore (e.g., CHEMSTRIP (RTM) LN; MULTISTIX (RTM) Reagent Strips), but preferably the sample is diluted to a certain volume and then the dilute sample is tested by dipping the reagent strip into the solution or by applying some of the solution to the reagent strip. The preferred commercially-available leukocyte esterase reagent strip for this procedure is CHEMSTRIP (RTM) LN which contains 5.8 µg of indoxyl carbonic acid ester (as described in the "CHEMSTRIP (RTM) LN" package insert). The present invention is performed utilizing diagnostic kits useful for detecting or evaluating the presence of periodontal diseases in humans. These kits comprise means for collecting a sample of fluid at one or more oral tissue sites (especially gingival sites) in a human being diagnosed and utilize an indoxyl carbonic acid ester for measuring the amount of leukocyte esterase. The phrase "means for collecting a sample of fluid", as used herein, means any device or apparatus or product which is useful for removing a sample of fluid (which may also include solid tissue matter) from the oral cavity for further analysis without adversely affecting the ability to detect the presence of leukocyte esterase activity in the sample of fluid collected. Non-limiting examples of such means are swabs, pipettes, syringes, absorbent tapes, absorbent gauzes, absorbent strips, scoops, suction bulbs, and aspirators. Preferred means are swabs and absorbent strips. The kits are manufactured such that the means for collecting a sample of fluid and the means for measuring the amount of leukocyte esterase are separate components in the kits, and these essential components plus any optional components to be utilized with the kits (e.g., test tubes for diluting samples in; bottles containing dilution fluid for diluting samples; instruction sheets; etc.) are combined into one package. An example of such a package is a box which is shrink wrapped with plastic. EXAMPLE I In Vitro Saliva Sample Assay Ten ml of a 0.9% saline solution is dispensed into a sterile container. Using this saline solution the patient (who has been instructed not to eat within the last hour) rinses his mouth for 30 seconds, after which time the rinse is returned to the container. This rinse is tested with the CHEMSTRIP (RTM) 9 leukocyte esterase diagnostic strip (sold by Bio-Dynamics, Indianapolis, Indiana) by briefly (no longer than 1 second) dipping the test strip into the saline rinse. The chemically impregnated patches on the test strip are totally immersed in the saline rinse before removal. Upon removal of the test strip from the solution, the edge of the test strip is drawn along the rim of the specimen container to remove excess rinse solution. Approximately 60 to 120 seconds after dipping the strip in the rinse solution, the test strip is read for leukocyte esterase activity by holding the strip in the same direction as when it was dipped into the saline and then carefully matching the patches with the proper color blocks provided on the color chart provided with the CHEMSTRIP (RTM) 9 product. Color changes that occur after 2 minutes from immersion are not of diagnostic value. Color changes that occur only along the edge of the test area are ignored (careful removal of excess saline should eliminate this effect). This diagnostic method correlates very well with the clinically observed gingival inflammation (GI) index. The GI index measurement is fully described in Loe, "The Gingival Index, the Plaque Index and the Retention Index Systems", J. Periodontology , 38 , pp. 610-616 (1967); and Silness and Loe, "Periodontal Disease in Pregnancy. I. Prevalence and Severity", Acta Odont. Scand. , 21 , pp. 533-551 (1963). EXAMPLE II In Vitro Gingival Site Assay In order to determine the relationship of gingivitis at specific gingival site to the presence of leukocytes at those sites, and to relate changes in gingival scores over time, the following diagnosis protocol is followed. Gingival inflammation is scored using the standard GI index with each tooth site classified as 0, 1, 2, or 3. After the patient's gingival sites are scored, the results are evaluated and three test teeth are selected per individual. Three teeth, with scores of 0, 1, and 2, are chosen in the upper maxilla for sampling. If all three scores are not available in the upper jaw, then mandibular teeth are selected. The tooth and gum at each site are briefly air-dried to remove saliva. A Type 4 Calgiswab R (sold by Spectrum Laboratories, Inc., Houston, Texas) swab is gently rolled in the crevicular margin for approximately 10 seconds. The swab is then immediately transferred to a plastic tube containing 1 ml of normal aqueous saline solution. The swab is gently swirled in the saline and the tube briefly agitated to disperse the collected material. After a minimum time of 30 seconds (preferably 10 to 20 minutes), a 20 µl aliquot of the sample solution is pipetted onto a CHEMSTRIP (RTM) LN leukocyte esterase diagnostic strip (sold by Bio-Dynamics, Indianapolis, Indiana). The color change is then recorded at 1 and 2 minutes after the solution is added to the diagnostic strip. Following this procedure it is determined that there is very good correlation between the clinically observed GI index and the CHEMSTRIP (RTM) LN evaluation. It is also determined that the CHEMSTRIP (RTM) LN diagnostic strip is predictive of the onset of gingivitis, and predictive of healthy improvement in diseased gingival sites during the course of therapy.

Claims: An in vitro diagnostic method for detecting or evaluating the presence of periodontal diseases in humans or lower animals, wherein said method comprises collecting a sample of saliva from the oral cavity or a sample from the gingival site to be diagnosed and measuring the amount of leukocyte esterase present in the sample utilizing an indoxyl carbonic acid ester which is hydrolyzable by leukocyte esterase to form indoxyl.

Inventors: WITT JONATHAN J, WITT, JONATHAN J.
Applicants: PROCTER & GAMBLE, THE PROCTER & GAMBLE COMPANY
Application year: 1988
Main classification: C12Q1
IPC codes: A61C19, C12Q1