DESC:TECHNICAL FIELD OF THE INVENTION
The present invention relates to a cost-effective and efficient kit for detecting bacterial vaginosis. More particularly, the present invention relates to a patient friendly, home test kit for detecting bacterial vaginosis.

BACKGROUND AND PRIOR ART OF THE INVENTION
The production of lactic acid has generally been considered to be the factor which allows Lactobacillus spp. to dominate the vaginal ecosystem. Lactobacillus species are usually predominant in vagina of healthy women and are believed to regulate the growth of other vaginal flora. However, lactobacilli have notably been observed to be absent on vaginal smears of women with bacterial vaginosis, and investigators have isolated Lactobacillus species less often from women with bacterial vaginosis (BV). More recently generated data suggests that H2O2 production by lactobacilli may be more relevant than lactic acid production. An absence of Lactobacillus species thus could allow an overgrowth of other pathogenic organisms, such as those found in high concentrations among women with bacterial vaginosis.

Bacterial vaginosis (BV) is one of the most common lower genital tract infections among women of childbearing age. BV, a condition characterized by reduced numbers of vaginal lactobacilli, has been associated with an increased risk of acquisition of HIV, while the absence of vaginal Lactobacillus spp. is associated with increased acquisition of HIV and gonorrhoea. Women vaginally colonized by H2O2-producing strains of lactobacilli are only half as likely to acquire bacterial vaginosis as women colonized by lactobacilli that do not produce H2O2.

BV is also associated with the development of an adherent polymicrobial biofilm containing abundant GardV bacteria (Gardnerella) on the vaginal epithelium. G.vaginalis is predominant in vaginal cultures from subjects with bacterial vaginosis. However, its diagnosis can be problematic.
At present, the presence of BV is detected on the basis of a clinical examination and Gram staining for the bacteria characteristic of the disease. Amsel method is still the gold standard for diagnosis of BV. The most widely accepted clinical criteria for diagnosis include three of the following four criteria:
• A vaginal pH of greater than 4.5,
• The presence of clue cells in the vaginal fluid,
• A milky homogeneous vaginal discharge, and
• The release of an amine (fishy) odour after the addition of 10% potassium hydroxide to the vaginal fluid.

However, interpretation of these criteria can be difficult and subjective. Detection of amine odour is observer dependent, with wide person-to-person variability in the ability to recognize the amine odour associated with bacterial vaginosis. Recognition of clue cells is an excellent predictor of bacterial vaginosis, but is likewise subject to variability depending on the quality of the microscope, the adequacy of the specimen, and the skill of the observer. Moreover, all these tests are to be carried out at a clinic for which women are usually reluctant to be tested.

A plethora of prior art literature has indicated attempts for either developing culture medium for detection of H2O2 production by Lactobacillus species or diagnostic kits for detection of bacterial vaginosis, a few of them are illustrated herein below along with the difficulties associated with them to perform tests at home and other unsatisfactory practices that do not yield fool-proof results.

Rabe and Hillier have, in the Journal of Clinical Microbiology, July 2003, p. 3260–3264, Vol. 41, No. 7 disclosed an optimized media for detection of hydrogen peroxide production by Lactobacilli species. The medium disclosed therein consisted of brucella agar base, 3,3’,5,5’-tetramethylbenzidine, horseradish peroxidase, starch, vitamin K, hemin, magnesium sulfate, manganese sulfate, and horse serum. However, such a medium for detection of bacterial vaginosis can be employed only in a laboratory set up, the results of which are not immediate and its application as a home diagnosis kit is impossible.

PCT Publication No.WO2018001215 with a Chinese priority patent application discloses a kit for detecting bacterial vaginosis, comprising: a detecting device, a color developing solution and a sample diluent, wherein the detecting device comprises a pH reaction pad, a hydrogen peroxide reaction pad, a leukocyte esterase reaction pad, and Sialidase reaction pad. Most importantly, this kit is concerned with clinical diagnosis of bacterial vaginosis and fails to provide a test for convenient home diagnosis. Further, it is observed from the disclosure of WO’215, that there is a need to dilute the sample isolated from a patient, therefore causing extended time durations for releasing test results and increasing the costs to develop such a kit. Additionally, WO’215, uses a color developing solution comprising several reagents. Manufacturing of such a complex device involves several steps and specialized setup, thus making the strip expensive to manufacture. Further, the presence of several reaction pads render the kit to be tedious and confusing for a technician/untrained person to carry out the test and later interpret the results, thereby generating a possibility of false results for BV. Moreover, this device is very expensive because of multiple tests and the related reagents involved. Further, this device of WO’215 requires a trained personnel to carry out the test and interpret the results. Additionally, the kit needs refrigeration for storage, which may not be necessarily available in rural areas.

Hence, it is important to have a home diagnosis test which gives the individual a clear idea about the occurrence or possibility of occurrence of bacterial vaginosis and makes it important for her to visit the doctor to obtain immediate treatment and relief. Such a home diagnosis test is required to be cost effective, so that it is accessible to masses irrespective of the societal differences in order to create awareness.

In light of the shortcomings posed by existing detection techniques and ill-equipped circumstances that women encounter, there is a need in the art for developing a cost effective product, particularly as a necessity in remote areas where female access to medical aid is rare and difficult and where there is a higher tendency to ignore the symptoms, till the final stage of the disease where complications associated with the bacterial vaginosis worsen.

OBJECT OF THE INVENTION
It is an object of the present invention to provide a cost-effective, user friendly and efficient kit for the diagnosis of bacterial vaginosis.

It is another object of the present invention to provide a home test kit for the diagnosis of bacterial vaginosis, mainly for the convenience of women in remote areas where the medical aid is not accessible.

It is yet another object of the present invention to provide a combined test indicating the pH and peroxide production levels to give a confirmation regarding the presence or absence of the BV infection.

SUMMARY OF THE INVENTION
The present invention provides a kit for detecting bacterial vaginosis comprising a carrier consisting of a pad for hydrogen peroxide detection and a pad to determine pH levels of vaginal fluid.

Accordingly, in an aspect, the present invention provides a kit for detection of bacterial vaginosis comprising;
(a) a swab or modified swab to collect vaginal fluid
(b) a carrier consisting of two reaction pads for determining peroxide activity and for indicating pH levels of vaginal fluid;
wherein the pad for determining peroxide activity (2) is impregnated with an enzyme and a chromogen such that the reaction of the enzyme with peroxide present in the vaginal fluid results in a color change; and
wherein the pad for determining the pH levels (1) is impregnated with a dye such that at pH levels lower than 4.5 of the vaginal fluid, a colour change is observed.

In keeping with the aforesaid aspect, the present invention provides a method of using the present kit for diagnosis of bacterial vaginosis comprising;
(a) taking the swab of the infected region or the region suspected to be infected to obtain a swab carrying the vaginal fluid,
(b) spreading the swab on the respective pad of the strips, allowing the reaction to take place for a duration of 10 minutes, and
(c) observing the color change to diagnose the occurrence of the disease.

In a further aspect, the present invention provides a cost-effective, user-friendly home test kit for the detection of bacterial vaginosis.

DETAILED DESCRIPTION OF DRAWINGS
Figure 1 is a diagrammatic representation of the device comprising a (1) pH pad and (2) a peroxide pad with immobilized reagents on a substrate.

DETAILED DESCRIPTION OF THE INVENTION
The invention will now be described in detail in connection with certain preferred and optional embodiments, so that various aspects thereof may be more fully understood and appreciated.

The H2O2 producing strains of lactobacilli are found in about 96% of women with normal vaginal microflora, but only in about 6% in women with bacterial vaginosis (BV), suggesting an important protective role against BV by the (H2O2)-producing lactobacilli. Therefore, the present diagnostic kit helps in detection of the occurrence of bacterial vaginosis by a dual process of detecting the change in pH levels, i.e. pH levels beyond 4.5 are characteristic of the occurrence of BV and change in color of the peroxide pad is indicative of absence of the disease.

In a preferred embodiment, the present invention provides a diagnostic kit for the detection of bacterial vaginosis by testing vaginal fluid for assessing the presence or absence of a vaginal infection comprising;
a carrier consisting of two reaction pads for determining peroxide activity and for indicating pH levels of vaginal fluid;
wherein the reaction pad for determining peroxide activity (2) is impregnated with an enzyme and a chromogen such that reaction of the enzyme with peroxide present in the vaginal fluid results in a color change; and
wherein the reaction pad for determining the pH levels (1) is impregnated with a dye such that at pH levels lower than 4.5 of the vaginal fluid, a colour change is observed.

While the embodiment herein above and in Figure 1 illustrates a carrier in the form of a holder strip, the carrier may, in alternate embodiments, be in the form of a disc or a stick or a card or other means provided that the carrier allows for the impregnation with an enzyme and a chromogen and fixation of the pH indicator to the padded portion of the strip to allow for ready application of a vaginal fluid.

In an embodiment, the present invention provides a carrier consisting of a holder strip having a length ranging from 50mm to 100mm, preferably 70mm to 90mm and most preferably 87mm, long enough for the user to hold the strip, without touching the reagent pads.

In another embodiment, the present invention provides a kit for detecting bacterial vaginosis comprising a carrier consisting of two reaction pads for determining peroxide activity and for indicating pH levels of vaginal fluid.

In accordance with the aforesaid embodiment, the reaction pad consists of a paper which is selected from a range of filter and chromatographic papers. The paper is selected based on its thickness, flow rate of the liquid through it, direction of the liquid flow (lateral or vertical) and pore size of the paper. Accordingly, the thickness of the paper ranges from 0.1 to 1mm, the flow rate of the liquid is in the range of 100mm-200mm/30 mins, wherein the flow direction should ideally be vertical, with pore size of the paper is in the range of 1 µm to 11 µm. The most suitable was found to be Whatman 3MM paper.

In another embodiment, the present invention provides Whatman 17CHR paper but the materials of the absorbent pads are restricted to chromatography paper.

In yet another preferred embodiment, the present invention provides a reaction pad consisting of a peroxide detection pad comprising a chromatography paper with a peroxidase and a chromogen adsorbed therein.

The pad for peroxidase activity carries peroxidase and a chromogen such that reaction of peroxidase and peroxide in the sample results in color change. The chromogen and peroxidase are taken in the ratio of 1:1.

The peroxidase enzyme is in a concentration ranging from 0.01 to 0.1% preferably 0.02%. The colour changes on the strip can vary with the varying reagents used. Most preferably, horseradish peroxidase (HRP) is used in the kit of the present invention.

The present invention provides chromogens selected from the group comprising tetramethyl benzidine (TMB) or 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS). More preferably TMB is used in the present invention.

Accordingly, the chromogen TMB, in the peroxidase activity pad is in a concentration ranging from 0.01 to 1%, preferably 0.1%. Further, ABTS when used is present in a concentration ranging from 0.0144mM to 3mM, preferably 2mM.

The peroxide strip consisting of ABTS, which gives a color change from colourless to pink, if peroxide is detected in the vaginal fluid sample, whereas presently, with the use of TMB, the color change is from colourless to blue, if peroxide is detected in the vaginal fluid sample. The chromogen is prepared in ethanol such that 0.1% of the chromogen is prepared in ethanol. The enzyme catalyst is prepared as 0.02% in buffer pH 6.

In a further embodiment, the present invention provides incorporating a stabilizer in a concentration ranging from 2% to 10% w/w in the peroxide pad.

Accordingly, several stabilizer solutions are available specifically to stabilize the peroxidase enzyme and its conjugates which are expensive chemical agents. In order to lower the cost, simple stabilizer solutions can be added directly to the solution of the enzyme, before coating on the paper, so as to stabilize the enzyme and further its conjugate after reaction. This allows the storage at room temperature which is otherwise not possible owing to enzyme instability. The stabilizers used in the present invention are selected form the group comprising polyvinyl alcohol, starch, polysaccharides and polyols.

A stabilizer allows the storage of the kit at room temperature, such that the reagents are stable and give reproducible results. A kit without stabilizer does not remain stable at room temperature and refrigeration is required for its storage. Another approach can be to directly insert the strip in the vagina such that the strip takes up the vaginal fluid and gives a color change. But for such a sterile strip is required. Since, the reagents are highly sensitive to heat, the strips cannot be sterilized. Hence, sterile swabs can be taken to finally transfer the swab liquid to the reaction pad and give the color change.

In yet another preferred embodiment, the present invention provides a reaction pad which serves as a pH value detection pad consisting of a filter paper comprising an azo dye which is adsorbed onto the paper and dried and gives a distinct color change with pH variations in the range of 3 to 6.

The vaginal pH of 3 to 3.5 indicates good vaginal health and a pH value to more than 4.5 is indicative of imbalance in the vaginal health and possibly infections. The color changing azo dyes giving a colour change in this pH range selected from the group consisting of bromocresol green, Alizarin red methyl orange, bromophenol blue and the like. Preferably, the use of bromophenol blue, gives a blue color at pH above 4.5 and decolourizes at lower pH.

The concentration of methyl orange and bromophenol blue is selected in a way that the color is intense enough to see a visual change on the reaction pad. The concentration range of the dye solution which is to be prepared is 0.5 to 5 mg/ml of the solution. The selected paper is dipped in the dye solution and dried. The number of dips should be restricted to 1, so as to ease the large scale manufacturing and shorten the duration of process. More number of dips may be required for lower concentrations of the solution. To avoid bleeding of the dye upon getting wet, fixatives layer can be added, which is basically a coating of polymeric solution. This would prevent the dye from bleeding from the strip, which may confuse the user to make out the colour of the reaction pad. The polymers are cellulose derivatives like hydroxyl propyl methyl cellulose, polyvinyl alcohol, hydroxyl propyl cellulose, polyvinyl acetate, polyethylene glycol. The concentration of the polymer ranges from 1% to 10% w/w, preferably 2% to 7% w/w, most preferably 5% w/w. The polymeric solution is coated on the substrate after the reagents are coated on it. This stabilizes the reagents and allows room temperature storage.
Accordingly, the dimension of the pads affixed onto the strip can be variable from 3*3mm to 6*mm, preferably 5*5mm. The more the surface area of the pad, the more amount of sample required to produce color since, the paper will absorb the sample solution. Hence, an optimum area of the sample pad is required, such as to note the color change distinctly. The pad for determining peroxidase activity is further stamped with a descriptive image for indicating of the diseased or non-diseased condition of the patient.

The substrates used as pads are available in varying thickness and flow rates. The ones which are selected are the ones, which have a good flow rate and are not too high thickness, since it affects the appearance of the strip

In yet another preferred embodiment, the present invention provides a diagnostic kit for the detection of bacterial vaginosis by testing vaginal fluid for assessing the presence or absence of a vaginal infection comprising;
(i) a swab or modified swab to collect vaginal fluid, and
(ii) a carrier consisting of two reaction pads for determining peroxide activity and for indicating pH levels of vaginal fluid;
wherein the reaction pad for determining peroxide activity is impregnated with an enzyme and a chromogen such that reaction of the enzyme with peroxide present in the vaginal fluid results in a color change; and
wherein the reaction pad for determining the pH levels is impregnated with a dye such that at pH levels lower than 4.5 of the vaginal fluid, a colour change is observed.

The present kit for the diagnosis of bacterial vaginosis provides for a swab or a modified cotton swab which is coated with an inert, non-irritant polymer that does not react with either the biological fluid or the diagnostic reagents. The present invention uses a cotton swab that can directly transfer the contents of the fluid isolated onto the absorbent pads without encountering issues of dilution of the sample.
It is important to note here that WO2018/001215 requires dilution of the sample taken from a patient. The present invention uses a cotton swab that can directly transfer the contents of the fluid isolated onto the absorbent pads without encountering issues of dilution of the sample.

The coating of the cotton swab prevents absorption of issues and the fluid isolated from the vagina before getting transferred to the absorbent pad. Therefore, a fool-proof test result is certain to be derived using the present diagnostic kit.

Accordingly, the present invention provides the cotton swab to be coated with hydrophilic polymers selected from the group comprising PVA- Polyvinyl alcohol or polyvinyl acetate, polyethylene glycol and hydroxy propyl methyl cellulose which are similar to those used for stabilization of the colour reaction pad for swab modification. The concentration used in the said purpose may be from 0.1% to 3%, more preferably 1% to 2%, most preferably 1.5%.The coating of the cotton swab prevents absorption of issues and the fluid isolated from the vagina before getting transferred to the absorbent pad. Therefore, a fool-proof test result is certain to be derived using the present diagnostic kit.

In one more preferred embodiment, the present invention provides a method of using the present kit for diagnosis of bacterial vaginosis comprising;
(a) swabbing the infected region or the region suspected to be infected to obtain a swab carrying the vaginal fluid;
(b) spreading the swab on the respective pad of the strips, allowing the reaction to take place,
(c) observing the color change to diagnose the occurrence of the disease.

Examples: Following examples are given by way of illustration therefore should not be construed to limit the scope of the invention.

Example 1: Diagnostic kit for the detection of bacterial vaginosis
A strip detecting the concentration of peroxide in the vaginal fluid has been designed as depicted in Figure 1. This strip consists of a reaction pad (2) consisting of an adsorbed peroxidase enzyme and a chromogen selected from the group comprising tetramethyl benzidine (TMB) or 2,2'-Azino-bis(3-ethyl benzothiazoline-6-sulfonic acid) diammonium salt (ABTS). The peroxidase enzyme on reacting with H2O2, if present in the vaginal fluid gives a colour change. The peroxide reaction pad consisting of ABTS, which gives a color change from colourless to pink, if peroxide is detected in the vaginal fluid sample, whereas, with the use of TMB, the color change is from colourless to blue, if peroxide is detected in the vaginal fluid sample. The chromogen is prepared in ethanol such that 0.1% of the chromogen is prepared in ethanol. The enzyme catalyst is prepared as 0.02% in buffer pH 6. The reaction pad (2) consisting of a peroxidase enzyme also consists of a stabilizer in a concentration of 5% w/w in the peroxide pad. The stabilizer is either polyvinyl alcohol, starch, polysaccharides or a polyol. The strip of the present invention also consists of a reaction pad which serves as a pH value detection pad (1) consisting of a filter paper comprising an azo dye which is adsorbed onto the paper and dried and gives a distinct color change with pH variations in the range of 3 to 6. The vaginal pH of 3 to 3.5 indicates good vaginal health and a pH value to more than 4.5 is indicative of imbalance in the vaginal health and possibly infections. To avoid bleeding of the dye upon getting wet, fixatives layer can be added, which is basically a polymeric solution. The diagnostic kit also consists of a cotton swab coated with 1% of the coating polymers selected from the group comprising Poly vinyl alcohol or polyvinyl acetate, polyethylene glycol and hydroxypropyl methyl cellulose.

Example 2
The diagnostic kit for the detection of bacterial vaginosis by testing vaginal fluid for assessing the presence or absence of a vaginal infection consists of a carrier consisting of two reaction pads for determining peroxide activity and for indicating pH levels of vaginal fluid, wherein the reaction pad for determining peroxide activity (2) is impregnated with a 0.02% horseradish peroxidase and TMB- 0.01% as a chromogen such that reaction of the enzyme with peroxide present in the vaginal fluid results in a color change. The peroxide activity pad also consists of a 5% w/w polyvinyl alcohol stabilizer solution combined with the peroxidase enzyme. The reaction pad for determining the pH levels (1) is impregnated with a 4 mg/ml bromophenol blue such that at pH levels lower than 4.5 of the vaginal fluid, a colour change is observed. In order to avoid bleeding of the dye upon getting wet, fixatives layer can be added, which is basically a coating of polymeric solution, i.e. PVA solution- 5%w/w.

Example 3
The diagnostic kit for the detection of bacterial vaginosis by testing vaginal fluid for assessing the presence or absence of a vaginal infection consists of a carrier consisting of two reaction pads for determining peroxide activity and for indicating pH levels of vaginal fluid, wherein the reaction pad for determining peroxide activity (2) is impregnated with a 0.02% horseradish peroxidase and 2mM of ABTS as a chromogen such that reaction of the enzyme with peroxide present in the vaginal fluid results in a color change. The peroxide activity pad also consists of a 5% hydroxypropyl methyl cellulose stabilizer w/w solution combined with the peroxidase enzyme. The reaction pad for determining the pH levels (1) is impregnated with a 4 mg/ml bromophenol blue such that at pH levels lower than 4.5 of the vaginal fluid, a colour change is observed. In order to avoid bleeding of the dye upon getting wet, fixatives layer can be added, which is basically a coating of polymeric solution, i.e. hydroxypropyl methyl cellulose solution- 5%w/w.
,CLAIMS:1. A diagnostic kit for the detection of bacterial vaginosis by testing vaginal fluid for assessing the presence or absence of a vaginal infection comprising;
a carrier consisting of two reaction pads for determining peroxide activity and for indicating pH levels of the vaginal fluid;
wherein the reaction pad for determining peroxide activity is impregnated with an enzyme and a chromogen such that reaction of the enzyme with peroxide present in the vaginal fluid results in a color change; and
wherein the reaction pad for determining the pH level of the vaginal fluid is impregnated with a dye such that at pH levels lower than 4.5 of the vaginal fluid, a colour is observed.
2. The diagnostic kit as claimed in claim 1, wherein the carrier is selected from the group comprising of a holder strip, a disc or a stick or a card.
3. The diagnostic kit as claimed in claim 1, wherein the reaction pad for determining the peroxide activity is impregnated with an enzyme peroxidase and a chromogen in a ratio of 1:1.
4. The diagnostic kit as claimed in claim 3, wherein the chromogen is selected from the group comprising tetramethyl benzidine (TMB) or 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS).
5. The diagnostic kit as claimed in claim 4, wherein TMB is in a concentration ranging from 0.01% to 1%.
6. The diagnostic kit as claimed in claim 4, wherein ABTS is in a concentration ranging from 0.0144mM to 3mM.
7. The diagnostic kit as claimed in claim 3, wherein the concentration of peroxidase is ranging from 0.01% to 0.1%.
8. The diagnostic kit as claimed in claim 1, wherein the reaction pad for determining peroxide activity and the reaction pad for determining the pH levels further comprises a stabilizer selected form the group comprising polyvinyl alcohol, starch, polysaccharides and polyols in a concentration ranging from 2% to 10% w/w.
9. The diagnostic kit as claimed in claim 1, wherein the reaction pad for determining the pH level of the vaginal fluid is impregnated with an azo dye which is adsorbed onto filter paper and gives a distinct color change with pH variations in the range of 3 to 6.
10. The diagnostic kit as claimed in claim 9, wherein the azo dye is selected from the group consisting of methyl orange, bromophenol blue, bromocresol green and Alizarin red.
11. The diagnostic kit as claimed in claim 9, wherein the concentration of the azo dye is prepared in a concentration ranging from 0.5 mg/ml to 5 mg/ml.
12. The diagnostic kit as claimed in claim 1, further consists a cotton swab coated with hydrophilic polymers selected from the group comprising PVA- Polyvinyl alcohol or polyvinyl acetate, polyethylene glycol and hydroxy propyl methyl cellulose in a concentration ranging from 1% to 3%.