MXPA99001460A - pH AND AMINE TEST ELEMENTS AND APPLICATIONS TO DIAGNOSIS OF VAGINAL INFECTIONS - Google Patents

Abstract

Tests for elevated pH and volatile amines in aqueous fluids are disclosed, including tests useful in the diagnosis of bacterial vaginosis and in other biological conditions. By using formulated indicators and indicators held in matrices that are permeable to gas but not to liquid, the tests provide clear and sharp transitions detectable by visual or machine-readable means rather than by subjective judgements such as small gradations in color or olfactory determinations. The tests lend themselves readily to iconic readouts of the test indications and to the inclusion of positive and negative controls.

Description

Elements and applications of the pH and amine test for the diagnosis of vaginal infections Field of the Invention The present invention relates to test device for clinical use, and in particular it relates to test devices for conditions that are characterized by an abnormal pH in an aqueous fluid, and by the conditions that are characterized hereby volatile amines. A particular area of interest for the use of these tests is the diagnosis of vaginal diseases.

Field of the Invention A recent study of bacterial vaginosis (BV) includes comparing the pH of vaginal fluids of women who were known to suffer from BV, with those women who were known not to have this disease. Gardner, H.L. et al. Am J. Obstet. Gynecol. 69: 962 (1955). It was determined that all women who tested positive for BV had a vaginal fluid with a pH greater than 4.5 and 91% of these women had a vaginal fluid with a pH greater than 5.0. In normal women (without the disease) that were part of the test, it was found that 92% had a vaginal pH between 4.0 and 4.7. The conclusion reached after the study was that the vaginal pH equal to or greater than 5.0, together with other clinical criteria indicated the present VB. Subsequent studies culminated in a report by Amsel, R. et al, Am J. Med 74: 14-22 (1983), which resulted in the reduction of the pH threshold for BV in 4.5 and established that the remaining criterion for Vaginal fluid homogeneity, breath test (treatment with alkali followed by an olfactory test to detect an amine smell) and the presence of key cells. This is known, commonly as Amsel clinical criteria for BV. The conclusion was based on a study group consisting of 397 women, of which 81% had a pH higher than 4.5, while only 23% of the women had a vaginal fluid with a pH greater than 4.5. In studies subsequent to the report by Amsel et al, the pH threshold was adjusted to 4.7. One of these studies was that of Holst EJ Clin Microbiol, 28: 2030-2039 (1990), in which 100% of the women diagnosed with positive VB according to the Amsel criterion were reported to have a pH of vaginal fluid greater than 4.7. Another of these studies, conducted by Eschenbach, D.A., Am. J. Obstet. Gynecol. 158 (4): 819-828 (1988), in which all 257 women studied had at least 20% of key cells, showed a pH in the vaginal fluid greater than or equal to 4.7, leading to the conclusion that the threshold value of 4.7 was better related to the clinical evidence of BV. Krohn, M.A. et al., J. Clin Microbiol. 27 (6): 1266-1271 (1989), also verified the correlation between the vaginal fluid pH threshold of 4.7 and the presence of key cells, and Holmes, KK, and their collaborators also confirmed that the limit of a pH in 4.7 as an indicator of VB - Holmes, KK, et al, eds Sexually Transmitted Diseases, McGraw-Hill, New York (1990), chapter 46: 527-545 (Holmes, KK et al) and Chapter 47: 547-559 (Hillier, SL et al). Colorimetry tests to determine an elevated pH of vaginal fluid use nitrazine yellow as an indicator. Nitrazine yellow is a monoazo dye that is bright yellow when the pH is 6.0 and bright blue when the pH value is 7.2 and has a midpoint with a greenish gray for a pH of 6. 6 However, in the range of interest for the evaluation of vaginal fluid, which is approximately 3.5 to 6.0, the change occurs in subtle progressions of greenish grays which are difficult to interpret. The limits or thresholds of pH are useful indicators in the diagnosis of a variety of biological conditions, both in humans and animals, and a large number of colorimetric indicators are known and commercially available. However, selecting the appropriate indicator is not always an easy task, and selections are often limited, particularly when a specific color change is desired and when stability of the indicator is a consideration. For example, whole cow's milk has a pH of 6.5 to 6.8 and has been reported (N.Z.J. Sci. Technol. 27: 258 (1945)) that cow's milk with a pH greater than 6.8 may indicate the presence of bovine mastitis. Next, the difficulties to detect the deviation of the pH, in such a small magnitude, using the indicators of conventional bands or paper strips will be discussed. A study specifically directed to bovine mastitis was reported in Dairy Sci. 68: 1263-1269 (1985). The purpose of the study was to determine if it is appropriate to use blotting paper impregnated with bromothymol blue, as pH indicator, to verify the pH of cow's milk, in the method to detect bovine mastitis. Milk was added to the paper treated with the indicator, and it was determined that the color of the pH was on a scale of 1 to 4, where 1 (pale green) was determined as normal (negative) and 2, 3 and 4 (an increase). from moderate green to dark greenish blue) was considered abnormal (positive). The pH of the milk was also determined electronically, with a carefully calibrated meter. The results of the test illustrated the difficulty in defining the color accuracy of the test area: the predictive value of a positive colorimetric test ranges from 49% to 52% (ie 51% to 48% of the results are false positives). As expected, the increase in the test measurements was compared with an increase in the severity of the mastistis as defined by the measurements of other diagnoses. However, in the milk of animals with less severe mastitis, the considerable overlap of the results "enhanced the possibility of an error in the interpretation of the indicator measurements." The wide variation of the pH of the milk (as determined electronically) ) behind each measurement with BTB (blue color Brom Timol) shows that the results of the indicator were not related to the pH. " The researchers stressed the importance of using color comparators that are as close as possible to the current pH test method. If the results of the pH tests of the milk with a colorimeter were not interpreted immediately, it was important to use comparators that were also moistened with milk. If the results of the pH test with a colorimeter are determined after the milk spots have dried, it was advantageous to use dry comparators. Back to bacterial vaginosis, the murmur test, which is one of the Amsel criteria, originated in a study conducted by Pheifer, et al, N. Engl J. Med 298, 1429-1434 (1978), reported the presence of a characteristic amine smell, similar to fish odor, after 10% KOH is added to the vaginal fluid of a woman with BV. The odor is caused by alkaline volatilization of amine salts in the vaginal fluid of a woman with BV. Unfortunately, the test is highly subjective, exposes the health of the person performing the test to a potential biological risk, and is susceptible to errors, since it is carried out in an object holder which, due to the ephemeral nature of the smell of the amine, should be placed directly under the nose and sniffed immediately after the addition of KOH. Alternatives to breath tests are analytical procedures such as high voltage electrophoresis (Chem, K.C.S. et al J. Clin Invest., 63: 828-835 (1979), thin layer chromatography (Chem, K.C.S. et al.

J. Infect Dis. 145: 337-345 (1982), and Sanderson, B.E. et al, Br. J. Vener. Dis. 59: 302-305 (1983), gas chromatography (Gravett, M.G. et al., Obstet, Gynecol., 67: 229-237 (1986) and Dravenieks, A. et al., J. Pharma. Se 59: 495-501 (1970), and high performance liquid chromatography (Cook RL et al, J. Clin Microbiol 30: 870-877 (1992) .These procedures, despite being more accurate and reliable than Breath test, they are expensive, they require more time and they are not suitable for on-site tests in a doctor's office or clinic.

The key cells or guide, which are another criterion of Amsel, are independently correlated with Vb, and in the hands of an expert microscopist are very sensitive and specific indicators of this infection. The key or guiding cells are squamous cells of the vaginal epithelium that are found in the vaginal fluid when there is a BV. The cells are covered with numerous bacteria, which gives them a granular appearance, and their edges darken or wrinkle due to the adherence of numerous bars or cocci. According to normal medical practice, the diagnosis of Vb is established when at least 20% of the epithelial cells detected are key or guiding cells. Homes et al, Sexually Transmitted Diseases, 2d de., McGraw-Hill, Inc. New York, 1990. The distinction between true key cells, in which a bacterium has completely obscured the edges of cells and cells that simply have a few attached bacteria require training and experience. A source of error is the similarity in appearance between the key cells and the trichomonas, white blood cells and other components of the vaginal fluid, which frequently result in an incorrect identification of these cells as key cells, and therefore give a False positive result. Another aspect is that when the key cells are present, they are often obscured by numerous components of the vaginal fluid, which means that the doctor does not consider all the key cells or quantify them at lower levels than the current ones. These can give a false negative as a result of the test. Therefore, it is highly desirable to have a distinctive analysis that is accurate and conveniently controlled and whose presence is related to the key cells.

Amine Test As in the pH test device of the present invention, the test device for the amine or test portion for the amine of the test device, which combines both tests, has a laminated structure. The individual sheets in the structure perform different and complementary functions, the sheets are coated collectively to provide an accurate and reproducible test, the results of which are read in their entirety by visual indications. The two main sheets are a gas-releasing sheet that has immediate access to the fluid specimen and a gas permeable indicator sheet but impermeable to the liquid specimens themselves or any of their components that are not in gaseous form. The gas-release sheet is a solid sheet of alkali that reacts with the amine salts in the specimen and converts them into volatile amines. Then, the volatized amines penetrate the gas permeable indicator sheet, where they cause a change in the color of the indicator. The terms "volatile amines" and "volatile amines" include amines that are only slightly volatile as well as those that are sufficiently volatile to escape into the atmosphere at substantial averages. The slightly volatile amines are those that only form a thin layer of gas on the liquid surface without significant amounts being released into the atmosphere. However, this thin layer of film is sufficient to penetrate the gas permeable indicator sheet. The selection of solid alkali for the gas-releasing sheet is not critical and may vary. In general, alkaline earth metal aluminates, carbonates and hydroxides can be used. Often, better results will be obtained using either sodium aluminate, sodium carbonate or magnesium hydroxide. Sodium aluminate is particularly preferred. The permeability only for gases in the gas permeable indicator sheet can be achieved in various ways, depending on the nature of the samples. A convenient method for aqueous specimens is the use of a porous hydrophobic polymer. Suitable polymers are those that are solid, insoluble in aqueous fluids, particularly vaginal fluid and take the form of layers easily, the coating or the sheet that does not dissolve, disperses in the form of particles, or disintegrates after coming into contact with these fluids. Examples of these polymers are ethyl cellulose, cellulose acetate and cellulose nitrate. Particularly preferred is ethyl cellulose. Alternatively, the indicator may be a water permeable, hydrophilic polymer that is covered with a hydrophobic sheet. Any indicator that changes color can be used, then it is exposed to the amines, and preferably to the amines in a fluid sample that can also be acidic. Bromocresol green is an example and can be used in the present, as well as in the pH test. Other examples are bromophenol blue, bromocresol purple, brorachlorophenol blue, nitrazine yellow, and several other indicators. The gas-releasing sheet and the gas-permeable indicator sheet are arranged in the test device so that the contact between the alkali in the gas-releasing sheet and the indicator in the gas-permeable sheet does not occur if there is no sample of gas. fluid. The arrangement is such as to allow the user a first contact of the gas-releasing sheet with the sample (preferably using an exchange, and then to put the gas-permeable indicator sheet in contact with the sample, so that the gas present in the sample penetrate into the gas indicator sheet, so that the indicator sheet, permeable to gas, can be the lower sheet in the device, being far from the surface in which the sample is applied, and the last sheet that touches the sample., this sheet can be applied on a base or support sheet, as in the pH indicator sheet. However, with the use of a polymer such as ethyl cellulose in the sheet matrix, it is structurally stable and reliable without the use of an underlying base sheet. In addition to the dispositions of these two sheets in relation to each other, the generation of volatile amines is improved by means of the devices and the penetration of these amines in the indicator, through the use of intermediate sheets that can also serve to define the geometric regions where A color change may occur, in addition the same type of information is provided by the pH test. As in the pH test, the color change in the amine test is preferably arranged in such a way as to cause the appearance of a particular geometric indicia, which is otherwise not visible. One means to achieve this is to restrict the access of the volatile amines to a geometric region defined in the gas permeable indicator so that the remaining regions of the sheet are still visible. These can be achieved by applying a gas guiding sheet directly on the gas permeable sheet, this gas guiding sheet is lightly transmissive to light or transparent and is made of a gas impermeable material, except in an opening or passage within its edges. Therefore, the volatile amines that are channeled through the opening, and the only portion of the permeable gas indicator sheet showing a color change is the area directly below the opening. There are several polymers that are examples of light transmissive materials which are also gas impervious, particularly polyamides, polyacrylates, sealants and varnishes. As an additional protection of the indicator in the gas permeable sheet of liquid amines, a sheet which is gas permeable, but impermeable to liquid can be placed directly on the gas channel sheet. For convenience, this protective sheet may be constructed of the same polymer used in the gas permeable indicator sheet. Again, ethyl cellulose is preferred. A second indicator region may be included in the amine test device to respond to the presence of a sample without considering whether there are volatile or volatilizable amines in the sample. As in the pH test, this second indicator region serves as an activated positive control of the sample, and can be applied as an additional sheet designated as a positive control sheet. The indicator in this sheet can be immobilized in a matrix that allows the penetration of the fluid sample, and the indicator itself can be any indicator that changes color after the application of the sample. The color change may be due to the non-volatile amines in the sample, or to the solid alkali in the gas-releasing sheet as the alkali is introduced into the sample with the use of an applicator brush. The immobilization matrix can be a hydrophilic polymer, examples of which were mentioned above when discussing preferences for the use of the pH indicator sheet in the pH test. The indicator can be the same used in the indicator sheet, permeable to gas, although any indicator that produces a color change after it comes into contact with the sample can be used. As in the pH test, the two indicators (ie, the indicator accessible only by volatile amines and the indicator on the positive control sheet that is suitable for both volatile and non-volatile amines, as well as the alkalinized sample) they are arranged in such a way that the two color changes are seen independently. Furthermore, it is preferred that the device include a third indicator region that remains visible but protected from the sample so that it does not change color under any circumstances. A) Yes, this region that does not change serves as a negative control background for the same reasons as the negative control in the pH test. In the same way, the preferred configuration of the indicators is in crossed bars forming a plus sign. Referring to Figures 3, 4a, and 4b, this can be achieved with the indicator sheet, permeable to gas 21 as the lower sheet, and the channeling sheet of gas 22 applied directly on it. An elongated gap 23 in the channel sheet divides the separated areas in two. The separation allows the passage of the volatilized amines and forms the vertical bar or line of a plus sign. A sheet with protective liquid barrier 24 (transparent) can also cover the channeling sheet of the gas 22, as well as the separation 23. Then, the positive control sheet 25 (which allows penetration by means of the liquid sample) is applied to the liquid barrier sheet 24, the positive control sheet has the shape of a bar that traces the horizontal portion of the plus sign. Finally, the gas release sheet 26 is applied as a separate layer in the device, either on the liquid protective barrier 24 or outside of all the sheets. The gas-releasing sheet in the arrangement shown in the drawings forms a ring around the horizontal bar or line of the positive control sheet, preferably without touching the bar. A small gap between the ring and the bar avoids a premature change of color in the bar. To use the amine test having the configuration described in the previous paragraph, a brush moistened with vaginal fluid is shaken around the gas-releasing ring 26, then passed through the entire circular area within the ring, so which comes into contact with the positive control horizontal bar 25 and the regions above and below it. The horizontal bar 25 will change color after coming into contact with any sample and any volatile amine released by the contact of the brush with the gas releasing ring penetrating the sheet of the liquid barrier 24 and passing through the separation of the vertical bar 23 in the channel sheet towards the indicator sheet, permeable to gas 21. The color change in the latter is restricted to the region of the vertical bar 27, while the rest of the indicator in the gas permeable sheet (the area 28 around the plus sign) is protected from the sample and therefore does not change color. Vaginal fluid samples that do not contain volatilized amines will produce a minus sign on the device (by causing a color change only on the horizontal bar 25), while samples that do not contain volatilizable amines will produce a plus sign (causing changes in color on both the horizontal bar 25 and the vertical bar 27). The indicators that were described in relation to both the pH test and the amine test are visual indicators, which produce a color change which is detectable by the naked eye. As alternatives, the present invention also contemplates indicators that can be read with the use of machines, or those that can be detected after they are excited or altered, such as fluorescent indicators. In preferred embodiments of the invention, the two tests are combined into a single test card 30 as shown in Figure 5. The two tests appear as separate areas 31, 32 on a surface of the card. Each one is highlighted in a dark circle 33, 34 to improve the user's visibility. The dark circle 34 surrounding the amine test zone may be wider because it contains the gas-releasing sheet that is needed to carry out the test. In addition, the card may contain information and instructions about use. The upper surface 35 of the card can be covered with a sheet for protection during storage and shipping, but is easily removed before use. The application of a biological sample in both test zones is easily achieved by means of a cotton swab or any brush capable of retaining a sample and applying it to the surface. In accordance with the present invention, a typical test card is approximately the size of a credit card, or approximately 2 inches (5.1 cm) by 3 inches (7.6 cm) and the test areas are circular, with diameters of approximately 0.25 inches (0.635 cm). The following examples are offered for illustration only. In these examples, the test elements according to the invention are used with the configuration shown in the figures. In the pH test element, the sheets mentioned below were used, using the reference numbers of Figure 1: base sheet 11: Mylar support with an ethyl cellulose coating applied as 15 percent on but of ethyl cellulose in n-propanol pH indicator sheet 12: EUDRAGIT RL PO and nitrazine yellow applied as a solution with the following composition: 25% EUDRAGIT RL PO, 0.34% yellow nitrazine, 8.6% sorbitol (wetting agent and permeabilizer, 70% aqueous solution), 10.0% 2-ethoxy ethanol, 12.4% deionized water, 43.66% 1-propanol (all by weight) the barrier sheet, channeling liquid 13: ethyl cellulose, applied as 15% of a solution in 30% n-propanol (all by weight) containing a separation 14 positive control sheet 15: EUDRAGIT RL PO with nitrazine yellow and bromocresol green, applied as a solution with the following composition: 22.8% of n- propanol, 12.40% of a deionized guide, 30.0% 2-ethoxyethanol, 8.60% sorbitol / 70% solution in water), 25.0% EUDRAGIT RL PO, 0.17% nitrazine yellow, 0.30% bromocresol green and 0.65% 2-sulfobenzoic acid anhydride (for resistance to humidity) (all by weight). In the test element of the amine, the sheets were as follows, using the references in Figure 3: the indicator sheet, permeable to gas 21: bromocresol green in ethyl cellulose, which is formed from a solution having the following composition: 1.8% of bromocresol green, 12% of ethyl cellulose and 86.2% of n-propanol (all by weight) the channeling sheet of gas 22: transparent polyamide, applied as a 20% solution of polyamide resin , 50% n-propyl acetate and 30% n-propanol (all by weight) liquid barrier 24: ethyl cellulose applied as a 10 weight percent solution of ethyl cellulose in n-propyl alcohol positive control sheet 25 : EUDRAGIT RL PO and bromocresol green applied as a solution with the following composition: 25% EUDRAGIT RL PO, 0.1% bromocresol green, 30.0% 2. ethoxy ethanol, 6.0% deionized water, 36.9% 1-propanol, 2.0% sulfobenzoic acid anhydride (all by weight) 26 gas-releasing sheet: sodium alumina applied as a solution of 28% sodium aluminate, 18% polyethylene, 3% maltodextrin and 51% deionized water (all by weight) To compare the test elements with elements and procedures of the prior art, two procedures were used. One consisted in the use of the bands for the COLORPHAST® pH test, elaborated by Em Science, Gibbstown, New Jersey, USA, which is sold for the exclusive use of physicians. The other procedure was the murmur test referred to above in the Background of the Invention, and consisting of applying a portion of the sample to an object slide, applying a drop of 10% KOH and then carrying out an olfactory determination. EXAMPLE 1 The pH test element according to the present invention was compared to a band for the COLORPHAST pH test, when performing pH tests on 607 samples of vaginal fluid. Of the 607 samples, 298 were negative (pH <; 4.7) according to the COLORPHAST test band, and 258 of these also gave negative according to the test element of the present invention. This represents the 86.6% coincidence in the negative result, with a standard deviation of 2.0% and a 95% confidence interval of 83.3%. Samples that tested positive according to the COLORPHAST test band (pH> 4.7) numbered 309, and of these, 263 were positive according to the test element of the present invention. This represents 85.1% coincidence in the positive result, with a standard deviation of 2.0% and a unilateral confidence interval of 81.8%. The average of coincidence was 85.8%, the standard deviation was 1.4% and the confidence interval 83.5%. EXAMPLE 2 In this example, the Amsel criterion was used to reconsider the samples that did not coincide in the tests carried out in Example 1. The four Amsel criteria are high pH, presence of amines by means of the murmur test, homogeneity in the vaginal fluid and presence of key cells. The theory behind this test is that the pH of the vaginal fluid was elevated due to a vaginal infection, then it would be expected that at least two of the other three Amsel criteria would be positive, to maintain the clinical diagnosis of bacterial vaginosis (BV). The 86 show discordant (where one pH test was positive and the other negative) were subjected to the three remaining Amsel criteria. Of the 40 specimens that gave negative results with the COLORPHAST test band and positive according to the test band of the invention, only two were clinically positive for BV by the Amsel criterion. Of the 46 samples that tested positive by the COLORPHAST test band and negative according to the band of the invention, 42 were clinically negative for BV by the Amsel criterion. This represents 98.5% of positive coincidence, 88.8% of negative coincidence and 93.1% of average coincidence, using the Amsel criterion to reconcile the discrepant results. EXAMPLE 3 This example compares the amine test element according to the present invention with that of the breath test. Both tests are carried out on a total of 617 vaginal fluid samples.

Of the total number of samples that undergo the test, 196 gave a positive result by means of the breath test, and of these, 166 also gave positive with the test element of the invention, representing 84.7% of coincidence, a standard deviation of 2.5% and a unilateral 95% confidence interval of 80.5%. Of the total number of tests, 421 gave negative results with the breath test, and of these, 385 also gave a negative result by means of the element of the invention, representing 91.4% of coincidence in negative results, a standard deviation of 1.4% and a unilateral 95% confidence interval of 89.2% The coincidence average was 89.3%, the standard coincidence 1.2% and the unilateral 95% confidence interval 87.3% EXAMPLE 4 The Amsel criterion was again used to reconsider the samples that did not coincide in the amine tests. Likewise, if the amines were detected because of a vaginal infection, it would be expected that at least two of the other three Amsel criteria would give positive results. The 66 samples of the amine test that did not match were tested following the three remaining Amsel criteria. The positive coincidence increased to 91.2%, the negative coincidence increased to 91.7% and the coincidence average was 91.6%. EXAMPLE 5 This example compares the combined pH and amine tests of the present invention with the combined results of the COLORPHAST test band and the normal murmur test (the reference tests). According to this test, the results using the present invention were considered positive when both tests gave positive results, and the result is considered negative in all other cases, that is, when either or both tests show negative results. Benchmark tests were designated in the same way -positive when both are positive, and negative when either or both are negative. Of the 604 vaginal fluid samples, 184 gave positive results according to the tests in reference, and 175 were positive according to the test of the invention. Similarly, 420 samples showed negative results according to the reference tests, and 429 were negative according to the tests of the invention. The disagreement occurs only in 45 cases. This represents 85.3% coincidence with a standard deviation of 2.6% and a unilateral 95% confidence interval of 81.0%, 95.7% of negative coincidence with a standard deviation of 1.0% and a unilateral 95% confidence interval of 94.1% and 92.5% confidence average with a standard deviation of 1.1% and a unilateral 95% confidence interval of 90.8% EXAMPLE 6 As with the individual test items, the discrepant results between the combined tests of the invention and the combined reference tests were reconciled using the Amsel criterion for a diagnosis of presumed BV. Of the 18 that were negative according to the combined reference tests, but were positive according to the tests of the invention, only two were clinically positive for VB according to the Amsel criterion. Of the 27 shown to be positive according to the reference tests but negative according to the tests of the invention, ten were clinically negative for VB according to the Amsel criterion. Thus, using the Amsel criterion to reconcile the discordant test results, the positive coincidence increased to 90.3%, with a standard deviation of 2.2% and a unilateral 95% confidence interval of 86.7%; the negative match increased to 96.3%, with a standard deviation of 0.9% and a unilateral 95% confidence interval of 94.8% and the coincidence average increased to 94.5% with a standard deviation of 0.9% and a unilateral 95% confidence interval of 93.0% EXAMPLE 7 This example illustrates the use of the present invention together with the two additional criteria of Amsel for the determination of the presence or absence of bacterial vaginosis (BV). As stated above, the diagnoses that use the Amsel method are based on four criteria: (1) the determination of the pH, (2) a breath test for the volatile amines, (3) an evaluation of the homogeneity of the vaginal fluid and (4) a microscopic examination of the vaginal fluid for the presence of key cells. According to traditional methods of evaluating test results, women who meet or cover at least three of the four criteria are considered to have positive BV. First, all the women in the study group were evaluated according to the four criteria, using the COLORPHAST pH test band for the pH test and the murmur test for the amines. Then the analysis was repeated in the same group using the same four criteria, but replacing the amine and pH tests of the present invention with the COLORPHAST and puff tests. The study group consisted of vaginal samples of 604 women. Based on the Amsel criterion using the COLORPHAST and breath tests for the determination of pH and amine, 161 (26.6%) of the women were considered to have positive BV and 443 / 73.4%) were classified as negative BV. Of the 161 positives, 153 were also classified as positive when the test was re-evaluated using the amine and pH tests of the present invention. Of the 443 negatives, 433 were also classified as negative when the criterion of the amine and pH tests of the present invention was re-evaluated. This represented 95.0% positive coincidence, with 1.7% standard deviation and a unilateral 95% confidence interval of 92.2%. similarly, the negative coincidence was 97.7%, with 0.7% standard deviation and a unilateral 95% confidence interval of 96.6%. The total coincidence was 97.0%, with 0.7% standard deviation and a unilateral 95% confidence interval of 95.9% and the kappa coefficient was 0.92, with 0.02 standard deviation and a unilateral 95% confidence interval of 0.90. The 18 samples that gave discordant results were reevaluated by the Gram stain test, according to which 7 or more were considered positive VB. Of the 10 samples that were VB negative by the Amsel criterion when the breath tests and COLORPHAST and VB positive were used according to the Amsel criteria when the amine and pH tests of the invention were used, 8 were clinically positive for VB according to the Gram spot test. Conversely, 8 of the samples that gave negative VB by the Amsel criterion when the breath and COLORPHAST and VB negative tests were used when the tests of the present invention were used, 1 proved clinically negative by the Gram test. Thus, when reconciling the discordant results with the Gram smear test, the positive coincidence increased to 95.8%, the negative coincidence increased to 99.5% and the coincidence average was 98.5% with a kappa coefficient of 0.96, a standard deviation of 0.01 and a unilateral 95% confidence interval of 0.94. EXAMPLE 8 This example illustrates the use of the amine and pH tests of the present invention as the test criterion for VB. The comparisons were made against the Amsel criterion (using the COLOPHAST band for the breath test for both pH and amines), and the Gram spot test was used again, to reconcile the discordant results, where the Gram stain yielded a result of 7 or more samples that were considered positive VB. For the pH and amine tests of the present invention, the diagnosis was considered positive only when both tests gave positive results. When one or both tests gave negative results, the diagnosis was considered negative. Of the 607 vaginal samples evaluated, 162 were positive by the Amsel criterion, and of these 162,139 also tested positive by the pH and amines tests according to the present invention (85.8%, standard deviation 2.7%, unilateral 95% confidence interval of 81.3%). Those tests that were negative according to the Amsel criterion were 445, and of these, 445, 408 (91.7%, standard deviation 1.3%, unilateral 95% confidence interval of 89.5%) were also negative according to the tests of amines and pH of the invention. This indicates a positive predictive value of 79.0% with a standard deviation of 3.1% and a unilateral 95% confidence interval of 73.9%, a negative predictive value of 94.7% with a standard deviation of 1.1% and a unilateral 95% confidence interval of 92.9%; and an efficiency of 90.1% with a standard deviation of 1.2% and a unilateral confidence interval of 05% of 88.1%. Then, the 60 discordant test results were reconciled by the Gram smear tests. Of the 37 that were VB negative by the Amsel criterion and positive according to the tests of the present invention, 24 were clinically positive according to the Gram's test. Of the 23 that gave positive VB by the Amsel criterion and negative by the tests of the invention, 4 were clinically negative by the Gram stain.

Thus, in reconciling the discordant results with the Gram stain test, 182 samples were positive VB by the Amsel criterion reconciled with Gram, and of these, 163 were positive by the two tests of the invention. Similarly, 425 samples showed negative results according to the criterion of Amsel reconciled with Gram, and of these, 412 were also negative according to the two tests of the invention. This increase in positive coincidence up to 89.6%, with a standard deviation of 2.3% and a unilateral 95% confidence interval of 85.8%; and the negative coincidence increased to 96.9%, with a standard deviation of 0.8% and a unilateral 95% confidence interval of 95.6%. These results indicate a positive, predictable value of 92.6% with a standard deviation of 2.0% and a unilateral 95% confidence interval of 89.4%; and a predictable negative value of 95.6%, with a standard deviation of 1.0% and a unilateral 95% confidence interval of 94.0% and an efficiency of 94.7%, with a standard deviation of 0.9% and a confidence interval of 95% unilateral 93.2%. EXAMPLE 9 This example illustrates the use of the amine tests of the present invention as a test for the key cells. Comparisons were made regarding the microscopic detection of the key cells, and the Gram stain test was used to reconcile the discordant results.

Microscopic examination for the key cells was considered positive when the key cells constituted more than 20% of the vaginal epithelial cells present in a vaginal fluid sample. As in the previous examples, the amines test performed according to the tests of the present invention were considered positive when the test element of the amines produces a bluer sign. Alternatively, the amine test according to the present invention was considered negative when the test element of the amines produces a minus blue sign. Of the 625 vaginal samples evaluated, 167 were interpreted as positive for the key cells with the use of the microscope. Of these 167 samples of positive key cells, 135 were positive according to the amine test of the invention (80.8%). The samples that were negative for the key cells with the use of the microscope were 458. Of these 458, 388 were negative according to the test of amines of the invention (84.7%). This indicates a positive coincidence of 80.8%, a negative coincidence of 84.7% and an efficiency of 83.7%. Then, the 102 discordant test results were reconciled with the Gram stain test. Of the 70 samples that were negative for key cells with the use of the microscope and positive with the amine test of the present invention, 36 were clinically positive for BV by Gram stain analysis. Thus, by reconciling the 70 samples that gave negative results for the key cells with the Gram stain test, 194 samples were positive for the key cells by the criterion of the microscope reconciled with the Gram stain, and 171 of these 194 shows were also positive according to the amine test of the invention. Similarly, of the 32 positive samples according to the microscope and negative criteria according to the amine test of the present invention, 9 samples were clinically negative for BV according to the Gram stain test. Thus, when reconciling the 32 positive test results of the key cells with the Gram stain, 431 samples gave negative results for the key cells according to the criterion of the microscope reconciled with the Gram stain, and 397 of these 431 show also gave negative according to the amine test of the invention. This increased the positive match to 88.1%, the negative match to 92.1% and the coincidence average to 90.9%. The foregoing is presented, mainly, for the purpose of illustrating the invention. It will be apparent to those skilled in the art that the configurations, dimensions, reagents and other materials, process steps and other parameters of the present invention can be modified or substituted in various ways, without departing from the spirit and scope of the present invention. .

Claims (86)

1. A method for immobilizing a pH indicator containing a phenol group that can be ionized and a negatively charged group on a solid matrix to prevent the indicator from spilling when it is wetted with a liquid aqueous sample, and to provide an indicator with a region of transition with a lower pH and lower range than the pH related to that of the indicator, when this is immobilized, this method comprises the formation of a solid composition comprising an indicator dispersed in a polymer containing quaternary amotino groups, practically in absence of covalent bonds between the indicator and the polymer. •2.

The method according to claim 1, wherein the pH indicator has a mid-range transition point and quaternary ammonium groups, which are present in the polymer, in an amount sufficient to reduce the mid-range transition point. at least 1.0 pH unit.

The method according to claim 1, wherein the pH indicator has a mid-range transition point, and the quaternary ammonium groups are present in the polymer in an amount sufficient to reduce the mid-range transmission point, approximately, from 1.0 pH unit to approximately 3.0 pH units.

The method according to claim 1, wherein the pH indicator has a mid-range transition point and in which the quaternary ammonium groups are present in the polymer in an amount sufficient to reduce the range transition point medium from about 1.5 pH units to about 2.5 pH units.

The method according to claim 1, wherein the pH indicator has a mid-range transition point and the quaternary ammonium groups are present in an amount sufficient to reduce the transition point of the middle range by approximately 2.0 units of pH.

The method according to claim 1, wherein the quaternary ammonium groups are present in an amount that supplies the polymer with an alkali value ranging from about 5 to about 50.

7. The method according to the re 1 , wherein the quaternary ammonium groups are present in an amount that supplies the polymer with an alkali value in a range from about 15 to about 40.

The method according to claim 1, wherein the quaternary ammonium groups are of trimethylammonium.

9. The method according to claim 1, wherein the polymer is an acrylic polymer.

The method according to claim 1, wherein the polymer is a copolymer of acrylic and methacrylic acid esters and the quaternary ammonium groups are trimethylammonium groups.

11. The method according to re 1, in which the negatively charged group of the oh-indicator is a member selected from a group consisting of sulfate and sulfonate groups.

The method according to claim 1, wherein the pH indicator is a member selected from a group consisting of bromophenol blue, bromochlorophenol blue, bromocresol blue, bromocresol purple, nitrazine yellow, bromothymol blue and bright yellow.

The method according to claim 1, wherein the pH indicator is nitrazine yellow.

14. A composition comprising a pH indicator containing an ionisable phenol groups and a negatively charged group dispersed in a solid polymer containing quaternary ammonium groups substantially in the absence of covalent bonds between the indicator and the polymer.

15. The composition according to claim 14 in which the pH indicator has a mid-range transition point and the quaternary ammonium groups are present in the polymer in an amount sufficient to reduce the mid-range transition point from about 1.0 unit. of pH up to about 3.0 pH units.

16. The method according to claim 14, in which the pH indicator has a mid-range transition point and in which the quaternary ammonium groups are present in the polymer in an amount sufficient to reduce the mid-range transition point from about 1.5 pH units to about 2.5 pH units.

The method according to claim 14, win the pH indicator has a mid-range transition point and the quaternary ammonium groups are present in an amount sufficient to reduce the transition point of the middle range by approximately 2.0 units. of pH.

18. The method according to claim 14, win the quaternary ammonium groups are present in an amount that supplies the polymer with an alkali value ranging from about 5 to about 50.

19. The method according to claim 14, wherein the quaternary ammonium groups are present in an amount that supplies the polymer with an alkali value ranging from about 15 to about 40.

The method according to claim 14, in that the quaternary ammonium groups are trimethylammonium groups.

21. The method according to claim 14, wherein the polymer is an acrylic polymer.

22. The method according to claim 14, wherein the polymer is a copolymer of acrylic and methacrylic acid esters and the quaternary ammonium groups are trimethylammonium groups.

23. The method according to claim 14, wherein the negatively charged group of the oh-indicator is a member selected from a group consisting of sulfate and sulfonate groups.

The method according to claim 14, wherein the pH indicator is a member selected from a group consisting of bromophenol blue, bromochlorophenol blue, bromocresol blue, bromocresol purple, nitrazine yellow, bromothymol blue and bright yellow.

25. The method according to claim 14, wherein the pH indicator is nitrazine yellow.

26. A test device for detecting a pH equal to or greater than the first selected transition point within the range of 4.6 to 4.8 in a sample of aqueous liquid, this test device comprises: a first polymer sheet, fluid permeable, solid hydrophilic and a negatively charged group, this polymer contains quaternary ammonium groups in an amount sufficient to immobilize the pH indicator against diffusion, when it is moistened with a sample of aqueous liquid and causes the indicator to change color at this first point of transition selected, - a second sheet of impermeable material covering the first sheet, the second sheet having an opening where the first geometric region of the first sheet is exposed; and a third sheet covers the second geometric region of the second sheet, this third sheet comprises a second pH indicator selected to change the color at a pH lower than the first selected transition point, at least about 0.7 pH units, this first and second regions are arranged in such a way that the color changes in them are visible independently.

27. The test device according to claim 26, wherein the second sheet is light transmissive.

The test device according to claim 26, wherein the second sheet is a polymer, light transmissive, hydrophobic.

29. The test device according to claim 26 in which the indicator in the third sheet is selected to undergo a color change at a pH of about 4.0 or less.

30. The test device according to claim 26, wherein the indicator in the third sheet is selected to undergo a color change at a pH of about 3.5 or less.

31. The test device according to claim 26, wherein the first and second geometric regions form two lines, straight, crossed so that a color change in the second geometric region, without there being a change of color in the The first geometric region forms a minus sign, while a change of color in both the first and second geometric regions forms a plus sign.

32. The test device according to claim 26, wherein the color changes in the first and second sheets are substantially equal color changes.

33. The test device according to claim 26, wherein the hydrophilic, permeable polymer is an acrylic polymer.

34. The test device according to claim 26, wherein the quaternary ammonium groups are trimethylammonium groups.

35. The test device according to claim 26, wherein the fluid permeable polymer, hydrophilic, is a copolymer of esters of acrylic and methacrylic acid and the quaternary ammonium groups are trimethylammonium groups.

36. The test device according to claim 26, wherein the pH indicator has a mid-range transition point, and the quaternary ammonium groups are present in the polymer in an amount sufficient to reduce the transition point of the medium range at least approximately 1.0 pH unit.

37. The test device according to claim 26, wherein the pH indicator has a mid-range transition point, and the quaternary ammonium groups are present in the polymer in an amount sufficient to reduce the range transmission point. medium, approximately, from 1.0 pH unit to approximately 3.0 pH units.

38. The test device according to claim 26, wherein the pH indicator has a mid-range transition point and in which the quaternary ammonium groups are present in the polymer in an amount sufficient to reduce the transition point of medium range from approximately 1.5 pH units to approximately 2.5 pH units.

39. The test device according to claim 26, wherein the pH indicator has a mid-range transition point and the quaternary ammonium groups are present in an amount sufficient to reduce the transition point of the middle range by approximately 2.0 pH units.

40. The method according to claim 26, wherein the quaternary ammonium groups are present in an amount that supplies the polymer with an alkali value ranging from about 5 to about 50.

41. The test device according to claim 26, wherein the quaternary ammonium groups are present in an amount that supplies the polymer with an alkali value ranging from about 15 to about 40.

42. The test device according to claim 26 in which the indicator of the third sheet comprises a mixture of nitrazine yellow and bromocresol green.

43. The test device according to claim 26 in which the permeable, hydrophilic polymer is a copolymer of esters of acrylic and methacrylic acid and the quaternary ammonium groups are trimethylammonium groups and the second lamina is ethyl cellulose.

44. The test device according to claim 26, wherein the third sheet comprises a hydrophilic polymer containing trimethylammonium quaternary groups.

45. The test device according to claim 26, further comprising a base sheet that is under the first sheet as a support thereof.

46. The test device according to claim 26, wherein the first and second regions are arranged so as to allow the third geometric region of the first sheet to be seen independently of the first and second regions and protects them from being wet with the liquid sample.

47. The test device for detecting salts of volatile amines in a liquid sample, this test device has an exposed surface and comprises: a gauge sheet, permeable to gas, made of a material that is permeable to gas but impermeable to liquids, with an indicator in it that undergoes a transition that can be detected after contact with the amines; and a sheet releasing the gaseous amine, comprising a solid alkali and forming part of the exposed surface leaving visible at least a portion of said indicator sheet which is gas permeable.

48. The test device according to claim 47, in which the indicator is defined as a first indicator, the test device further comprises an indicator sheet, permeable to the liquid, which is impregnated with the second indicator, which presents a Transition that can be detected after coming into contact with an alkali, the first and second indicators are arranged so that the transitions can be detected independently.

49. The test device according to claim 48, further comprising a sheet channeling the gas interposed between the gauge sheet, permeable to the gas and the indicator sheet permeable to the liquid, the channeling sheet of the gas has a first region geometric defined in it, which is permeable to gas, the rest of this channel sheet is impermeable to gas; and the gas permeable indicator sheet covers the second geometric region of the channel sheet, the first and second geometric regions are visible independently.

50. The test device according to claim 49, wherein the first and second geometric regions are arranged to leave the third geometric region, of the gas permeable indicator sheet, visible independently of the first and second regions and protected against any color change that results from the application of the liquid sample on the test device.

51. The test device according to claim 47, wherein the indicator is a visual indicator that changes color after coming into contact with gaseous amines.

52. The test device according to claim 48, wherein the first and second indicators are visual indicators that change color after coming into contact with amines.

53. The test device according to claim 47, wherein the material of the gas permeable indicator sheet is ethyl cellulose.

54. The test device for detecting salts of volatile amines in a sample of aqueous liquid, this test device has an exposed surface and comprises: a gas-permeable indicator sheet, made of a gas permeable material but impervious to liquids, impregnated with a first indicator that changes color after coming into contact with amines; a sheet that serves as a barrier for the liquids, gas channeler, which covers the gas permeable indicator sheet, said gas channel sheet has a first geometric region defined therein, and a first geometric region that is impermeable for aqueous liquids but permeable for gas, the rest of the sheet that serves as a barrier for aqueous liquids, gas channeler, is impermeable both to aqueous liquids and gases; an indicator sheet, which is permeable to aqueous liquids, which covers a second geometric region of the sheet that serves as a barrier for the liquids, channeling the gas, the indicator sheet, permeable to the liquid comprises a second indicator that changes color afterwards to come into contact with the alkali, the first and second geometric regions are arranged so that the color change in them can be seen independently; and the gaseous amine-releasing sheet forms part of the exposed surface and does not cover or overlap with the second geometric region, the gaseous amine-releasing sheet, comprising a solid alkali.

55. The test device according to claim 54, wherein the sheet which serves as a liquid barrier, channeling the gas, is transmissive of light.

56. The test device according to claim 54, in which the sheet serving as a gas channeling barrier comprises two sublaminae, one is gas channeler, that is, it is impermeable to gas except in one opening through the gas. where the gas inlet is channeled, this first geometric region is defined by the opening, and the other sub-plate serves as a barrier for liquids, so it is impermeable to liquids but permeable to gases.

57. The test device according to claim 54, wherein the first and second geometric regions form two straight lines so that the color change in the second geometric region without a color change occurring in the first geometric region forms a minus sign, while the color change, both in the first and in the second geometric regions form a plus sign.

58. The test device according to claim 54, wherein the gaseous amine-releasing sheet surrounds the first and second regions.

59. The test device according to claim 54, wherein the solid alkali of the gaseous amine releasing sheet is a member selected from a group consisting of alkali metal and alkali metal aluminates, carbonates and hydroxides.

60. The test device according to claim 54, wherein the solid alkali of the gaseous amine sheet is a member selected from a group consisting of sodium aluminate, sodium carbonate and magnesium hydroxide.

61. The test device according to claim 54, wherein the solid alkali of the amine-releasing sheet is sodium aluminate.

62. The test device according to claim 54, wherein the color changes in the gas permeable indicator sheet and the liquid permeable indicator sheet are substantially identical in color changes.

63. The test device according to claim 54, wherein the first indicator and the second indicator are equal.

64. The test device according to claim 54, wherein the first indicator and the second indicator are both bromocresol green.

65. The test device according to claim 54, wherein the permeable indicator sheet is a hydrophilic polymer impregnated with the second indicator.

66 The test device according to claim 56, wherein the sub-plate serving as a liquid barrier and this material of the gas-permeable indicator sheet are hydrophobic polymers.

67. The test device according to claim 56, wherein the sublamina which serves as a barrier for liquids and the material of the indicator sheet, permeable to gas, are hydrophobic polymers and the indicator sheet, permeable to liquid, consist of a hydrophilic polymer impregnated with a second indicator.

68. The test device according to claim 54, wherein the material of the indicator sheet, permeable to gas, comprises ethyl cellulose.

69. The test device according to claim 56, wherein the sublamina which serves as a barrier for the liquids and the material of the gas-permeable indicator sheet, are made of ethyl cellulose and the liquid-permeable indicator sheet comprises a Acrylic polymer containing quaternary ammonium groups.

70. The test device according to claim 56, wherein the sublamina which serves as a barrier for the liquids and the material of the gas-permeable indicator sheet are of ethyl cellulose and the gas-permeable indicator sheet is of a copolymer of esters of acrylic acid and methacrylics containing trimethylammonium groups.

71. The test device for analyzing a liquid sample for both (a) a pH equal to or greater than the first transition point, selected within a range of 4.6 to 4.8 and (b) for the presence of volatile amines salts , and this test device comprises first and second test zones, the first test zone consists of: a pH indicator sheet made of a polymer, permeable to fluids, hydrophilic, impregnated with a first indicator containing a phenol group which can be ionized and a negatively charged groups, this polymer contains quaternary ammonium groups in an amount sufficient to immobilize the first indicator against diffusion when moistened with a liquid sample and to cause this indicator to change the color at the first point of selected transition; the liquid channeling sheet made of a liquid impervious material, which covers the pH indicator sheet, the liquid channel sheet has an opening through which a first geometric region of the pH indicator sheet is exposed; and a first positive control sheet covering a second geometric region of the liquid channel sheet, the first positive control layer comprises a second indicator selected to change the color at a pH lower than the first selected transition point, At least about 0.7 pH units, the first and second geometric regions are arranged so that the color changes so that it is visible independently; and the second test zone comprising: an indicator sheet, permeable to gas, made of a gas permeable material but impermeable to liquids, impregnated with a third indicator that undergoes a color change after coming into contact with the amines; the gas-guiding sheet, which is below the gas-permeable sheet, this gas-guiding sheet has a third geometric region defined therein, which is impermeable before liquids but is permeable to gas, the rest of the channel sheet The gas is impermeable to both liquids and gases; a second positive control sheet covers a fourth geometric region of the gas channeled sheet, the second positive control sheet comprises a fourth indicator that changes color after coming into contact with an alkali, the third and fourth geometric regions are disposed of such that it detects the transitions and presents them in such a way that they are visible independently; and a gas-releasing sheet forming a fifth geometric region of this test device, in which at least some portions of the first, second, third or fourth geometric regions are left, without being covered by the gas release sheet, This gas-releasing sheet comprises a solid alkali.

72. The test device according to claim 71, wherein the gas guiding sheet is impermeable to gases, except in the opening through which the gas penetrates, the third geometric region defined by this opening, and the test device that also comprises a sheet that serves as a barrier for liquids, is impermeable to liquids but permeable to gases, which covers the channeling sheet of gas.

73. The test device according to claim 71, wherein the first and second geometric regions form straight, crossed lines, so that the color changes in the second geometric region, without changing in the first geometric region, forming a minus sign, while there is a change of color in both the first and second geometric regions and forms a plus sign, and in which the third and fourth geometric regions form two straight lines crossed so that the color changes in the fourth geometric region without there being a change of color in the third geometric region forming a minus sign while changing the color in both the third and fourth geometric regions form a plus sign.

74. The test device according to claim 71, wherein the gas-releasing sheet surrounds the third and fourth geometric regions.

75. The test device according to claim 71, wherein the indicator on the first positive control sheet is selected to perform a color change at a pH of about 3.5 or less.

76. The test device according to claim 71, wherein the polymer, permeable for the hydrophilic liquids, of the pH indicator sheet, is an acrylic polymer having trimethylammonium groups.

77. The test device according to claim 71, wherein the fluid permeable, hydrophilic polymer of the pH indicator sheet and the first and second sheets for the positive control comprises acrylic polymers having trimethylammonium groups.

78. The test device according to claim 72, wherein the liquid channel sheet, the liquid barrier sheet and the pH indicator sheet have ethyl cellulose.

79. The test device according to claim 71, wherein the first indicator is nitrazine yellow.

80. The test device according to claim 71, wherein the first indicator is nitrazine yellow and the quaternary ammonium groups are present in an amount sufficient to cause the nitrazine yellow to change color to a pH of about 4.7. .

81. The test device according to claim 71 in which the first and second regions are arranged to allow the sixth geometric region of the pH indicator sheet to be visible independently of the first and second geometric regions and be protected from get wet with the sample of liquid.

82. The test device according to claim 71, in which the third and fourth geometric regions are arranged to allow the seventh geometric region of the permeable indicator sheet to be visible independently of the third and fourth geometric regions and to be protected against any color change resulting from the application of a liquid sample containing the amines to be analyzed.

83. The test device according to claim 71, wherein the first and second geometric regions are arranged to allow the sixth geometric region of the pH indicator sheet to be visible independently, to the first and second geometric regions, and the Third and fourth geometric regions are arranged to let the seventh geometric region of the indicator sheet, permeable to gas, be visible independently of the third and fourth geometric regions, the sixth and seventh geometric regions are protected so that any color change that results from the application of the liquid sample, which contains the amines to be analyzed.

84. The method for the diagnosis of bacterial vaginosis in a patient, by analyzing a sample of the patient's vaginal fluid, the method includes the determination whether the vaginal fluid is positive in both of the following cases: (1) a test for a pH equal to or greater than the selected transition point within the range from 4.6 to 4.8, carried out by applying this sample to a compound consisting of a first pH indicator, which contains a phenol ionizare groups and a negatively charged group, dispersed in a hydrophilic fluid permeable polymer, containing quaternary ammonium groups in sufficient quantities to immobilize the pH indicator against diffusion when wetted with a liquid sample and to cause the indicator to change color at this first point selected transition; and (2) a test for the salts of volatile amines, which is carried out by applying this sample to a solid alkali and a gas-permeable indicator sheet made of a gas-permeable but liquid-impermeable material, impregnated with an indicator that undergoes a detectable transition after it comes in contact with the amines.

85. The method for the diagnosis of bacterial vaginosis in a patient, when analyzing a vaginal fluid sample from a patient, the method involves the determination of a positive vaginal fluid test, at least in three of the following four tests (1) a test for a pH equal to or greater than the selected transition point within the range of 4.6 to 4.8, which is carried out when applying the sample to a compound comprising a first pH indicator containing a group phenol that can be ionized and a negatively charged group, dispersed in a hydrophilic fluid permeable polymer, containing quaternary ammonium groups in an amount sufficient to immobilize the pH indicator against diffusion when wetted with a liquid sample to cause that the indicator changes color at the first selected transition point; (2) a test for the salts of volatile amines, which is carried out by applying a sample to a gas permeable and solid alkali indicator sheet of gas permeable material but impervious to liquids, impregnated with an indicator that he undergoes a transition that can be detected when he comes in contact with the amines; (3) a test for vaginal fluid homogeneity; and (4) a test to detect the presence of key or guide cells.

86. The method to analyze a sample of vaginal fluid, to verify the presence of key cells, the method includes the application of the sample to a gas-permeable and solid alkali indicator sheet, of gas permeable material but impervious to liquids , the gas permeable indicator sheet impregnated with an indicator that undergoes a transition that can be detected after coming into contact with amines, the penetration of the amines into the gas permeable indicator sheet related to the present of key cells in the sample.