Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Reexamination Certificate
2000-02-08
2001-07-03
Leary, Louise N. (Department: 1623)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
C435S004000, C436S111000
Reexamination Certificate
active
06255066
ABSTRACT:
FIELD OF THE INVENTION
The invention pertains to a method for screening for bacterial vaginosis and a diagnostic kit for use therein. More generically, the invention pertains to a method for detecting the presence of an amine in a sample of fluid, a diagnostic kit for use therein, as well as a method for making a dye, especially 2,6-dichloroindophenol.
BACKGROUND OF THE INVENTION
Vaginitis is the most common gynecological problem in adult women. Infectious vaginitis presents itself in three primary forms: (a) bacterial vaginosis, (b) candidal vaginitis or “yeast”, and (c) trichomonas vaginitis or “trich.” Bacterial vaginosis, which affects up to 25% of American women in the normal clinical populations, is nearly twice as common as Candida and is, in fact, the most common form of vaginal infection. Bacterial vaginosis is caused by a replacement of the normal vaginal flora with facultative anaerobic bacteria, primarily
Gardnerella vaginalis.
Unfortunately, the symptoms of bacterial vaginosis are non-specific or non-existent and differential diagnosis is problematic.
Complications associated with bacterial vaginosis represent a major health care cost burden. For example, obstetric complications of bacterial vaginosis include (1) preterm labor/birth, (2) low birth weight babies; (3) premature rupture of the amniotic membranes or PROM; (4) amniotic fluid infections; (5) postpartum endometritis, and (6) chorioamnionitis. Preterm/low birth weight babies is the second leading cause of infant mortality, next to birth defects. Also, bacterial vaginosis is suspected of being one of the many causes of cerebral palsy. In addition, gynecologic complications of bacterial vaginosis include (1) postoperative infections; (2) pelvic inflammatory disease (PID); (3) abnormal cervical cytology, (4) increased susceptibility to sexually transmitted diseases (STDs), and (5) posthysterectomy infections. STDs such as chlamydia, herpes, syphillis, gonorrhea, and trichomoniasis also cause potential harm to the fetus. Furthermore, a Swedish study reported in
Acta Obstetricia et Gynecologica Scandinavica,
73:586-588 (1994) suggests that bacterial vaginosis may potentially be a cofactor with human papilloma virus in the development of cervical intraepithelial neoplasia (CIN), a precursor of cervical cancer.
While the rapid, accurate diagnosis of bacterial vaginosis is critical to an effective treatment decision and can minimize serious complications and costs, recent studies show that many women with bacterial vaginosis incorrectly self-diagnose their symptoms, mistaking them for yeast infections. The consensus among experts today is that proper differential diagnosis of vaginitis is essential for any OB/GYN practice, and that routine screening for bacterial vaginosis may become increasingly appropriate. Northern Europe has already implemented this approach and now conducts bacterial vaginosis tests in conjunction with annual Pap smears. Furthermore, since self-obtained vaginal swabs have been shown to be reliable specimens for use in the diagnosis of bacterial vaginosis, experts agree that home diagnostics may be the wave of the future.
SUMMARY OF THE INVENTION
One embodiment of the present invention is a screening technique for bacterial vaginosis that can be used at home or in a doctor's office. The bacterial vaginosis screening technique is directed to detecting the presence of one or more amines (e.g., putrescine (a.k.a. 1,4-diaminobutane or tetramethylenediamine) and cadaverine (a.k.a. 1,5-diaminopentane) which are known to occur at significantly elevated levels in Gardnerella-related vaginitis) in a sample of vaginal fluid and comprises the steps of (a) combining the sample of vaginal fluid with phenol (or any developer reagent selected from the group consisting of phenol, derivatives thereof, and mixtures thereof) to form a first reaction medium; (b) combining the first reaction medium with sodium hydroxide and/or potassium hydroxide (or any other water soluble base) and sodium hypochlorite (or any other halogen-containing oxidizing agent) to form a second reaction medium having a pH of at least about 9.5; and (c) observing the color of the second reaction medium (preferably to determine the presence of a 2,6-dichloroindophenol salt), where the concentration of the halogen-containing oxidizing agent in the second reaction medium is about 0.12 or greater (on a molar basis) than the concentration of the developer reagent in the second reaction medium. If the screening technique detects the presence of amines, the patient should be seen by a physician or at a clinic to have a microbial culture of her vaginal fluid grown out to determine the exact source of the amine-producing organism in order for a physician to prescribe an appropriate treatment plan.
A second and broader embodiment of the invention encompasses employing the foregoing method to detect the presence of an amine in any sample of fluid.
A third embodiment of the invention is a diagnostic kit suitable for use in the above described procedures. The diagnostic kit comprises (a) the developer reagent; (b) the water soluble base; and (c) the halogen-containing oxidizing agent, where (i) the concentration of the halogen-containing oxidizing agent in a reaction medium formed by combining the developer reagent, the water soluble base, and the halogen-containing oxidizing agent is about 0.12 or greater (on a molar basis) than the concentration of the developer reagent in the reaction medium and (ii) the pH of the reaction medium is at least about 9.5.
In a fourth and broader embodiment, the invention encompasses a method for preparing a dye which comprises the steps of: (a) reacting the developer reagent with an amine to form a first reaction medium; and (b) combining the first reaction medium with the water soluble base and the halogen-containing oxidizing agent to form a second reaction medium having a pH of at least about 9.5, where the concentration of the halogen-containing oxidizing agent in the second reaction medium is about 0.12 or greater (on a molar basis) than the concentration of the developer reagent in the second reaction medium.
The above-summarized embodiments of the invention as well other embodiments thereof are described in more detail below.
DETAILED DESCRIPTION OF THE INVENTION
One method for screening for the presence of bacterial vaginosis in accordance with the present invention entails taking a sample of vaginal fluid. This can be accomplished, for example, by a vaginal swab. The sample of vaginal fluid is then contacted with a developer reagent to form a first reaction medium. The first reaction medium is next combined with a solution comprising a water soluble base and a halogen-containing oxidizing agent to form a second reaction medium, which typically has a pH of at least about 9.5. After a period of time (such as after a lapse of about 5 to 10 minutes) the color of the second reaction medium is observed. Alternatively, the sample vaginal fluid can be initially combined with a solution that comprises the developer reagent, the water soluble base, and the halogen-containing oxidizing agent or the sample of vaginal fluid can be first mixed with developer reagent and then mixed sequentially with the water soluble base and the halogen-containing oxidizing agent in any desired order.
The developer reagent employed in the bacterial vaginosis screening technique is generally selected from the group consisting of phenol, phenol derivatives (e.g., 2-phenylphenol, 3-phenylphenol, 4-phenylphenol), and mixtures of thereof. Phenol is the preferred developer reagent.
The amount of developer reagent employed in the methodology of the present invention should be sufficient to react with enough of any amine present in the sample to obtain a positive result. Generally, the developer reagent is used in an amount such that the concentration of the developer reagent present in a reaction medium formed by combining the sample of fluid, the developer reagent, the water soluble base, and the halogen-containing oxidizing agent (and
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