Vaginosis is a widely prevalent syndrome. According to a study conducted by the Indian Council of Medical Research (ICMR), the prevalence of this syndrome is about 30% in women attending the antenatal clinics in urban Delhi [1]. Bang reported in Lancet its incidence to be 50% in rural Maharashtra [2]. Its incidence was 80% in slums of Delhi in a survey conducted by us along with Safdarjung Hospital and Population Foundation of India [3]. It is reported as a public health problem of concern in USA [4]. Its prevalence in the United States is estimated to be 29.2% in women of 14-49 age [5].Vaginosis is expressed as abnormal vaginal discharge, fishy odour and the pH of vagina above 5.0 with absence of normally dwelling Lactobacilli. The present treatment for Bacterial vaginosis employs various antibiotics which have different spectrum of activity with a cure rate at 4 weeks of 60%–70% [6]. The antibiotic Metronidazole usually prescribed is not pleasant to take for many women who experience nausea, dizziness, drowsiness, vomiting, headache and metallic taste [7]. The recurrence of vaginosis is frequent in women treated with antibiotics.

As alternate to antibiotics, we considered making a formulation employing traditionally used herbs of age old known properties. This polyherbal microbicide named as BASANT was formulated with 95% pure diferuloylmethane {(1E, 6E)-1, 7-bis (4-hydroxy-3- methoxyphenyl)-1, 6- heptadiene-3, 5-Dione} (Curcumin) from Curcuma longa, purified extracts of Emblica officinalis, Neem (Azadirachta indica) leaves, and Aloe vera (Aloe barbadensis). These ingredients were dispensed along with pharmacopoeially approved excipients: citric acid, sorbitol, microcrystalline cellulose, sodium starchglycolate, starlac, Crospovidones and sodium alginate as a lubricating agent.

BASANT was formulated as both a cream and as powder encapsulated in easily dispersible cellulose capsules. It was tested & found effective against WHO strains and clinical isolates of Neisseria gonorrhoeae, including those resistant to penicillin, tetracycline, nalidixic acid and ciprofloxacin [8]. BASANT has also pronounced inhibitory action against Candida glabrata, Candida albicans and Candida tropicalis isolated from women with vulvovaginal candidiasis, including three isolates resistant to azole drugs and amphotericin. Table 1 Summarizes the variety of genital pathogens against which this microbicide was tested & found to exercise inhibitory action. At the National Cancer Institute, National Institutes of Health (NIH), Dr. John Schiller observed that BASANT inhibits the entry of HPV16 in Hela cells [6]. Prof. Rajyashree Sharma Head of the Department of Obstetrics & Gynecology at Medical College, Aligarh found that 30 daily intravaginal intakes of BASANT capsules caused the elimination of HPV 16/18 from the infected cervical cells of women at early stages of Carcinoma of Cervix patients. Their Pap smear also became normal. At the National Institute of Pathology, New Delhi, Prof. Aruna Mittal found that BASANT exercised an inhibitory action on Chlamydia trachomatis whether tested against the free bacteria or lodged in cells [9]. More recently, studies carried out at the University of Oregon, USA by Prof. Manoj Pastey reported BASANT to be effective against both CCR5 and CCR4 tropic HIV 1 lab-adapted strains and primary isolates belonging to different clades [10]. Dr. Smita Kulkarni at the National Institute of Virology Pune has found that BASANT inhibits various HIV-1 subtypes at non cytotoxic concentration. It was also found to inhibit HIV-1 replication in the Epi Vaginal explants model. Thus this Polyherbal microbicide has a wide spectrum action on a variety of genital pathogens, including HIV.

Table 1: BASANT inhibits the following genital pathogens.

Human female vagina has some interesting characteristics differing from the rest of the body. Healthy vagina has an acidic pH of about 4.5 whereas the normal pH of the rest of the body is 7.4. Acidic pH is generally protective to discourage growth and colonization of a large number of microorganisms. The acidic pH of vagina is caused and maintained by Lactobacilli strains which co-exist along with the endometrial cells of healthy vagina. To learn more about these indwelling bacilli which confer an important health benefit, we isolated from 80 healthy women with their written consent, Lactobacilli resident in the vagina. These were characterized on the basis of Genus, Group and Species specific PCR’s along with Random Amplified Polymorphic DNA (RAPD) and 16s rDNA analysis [11].The most frequent Lactobacilli strains found in vagina of women in Delhi belong to Group IV (L.reuteri, L.fermentum, L.salivarius, L. plantarum) in contrast to Group II (L.crispatus, L.jensenii, L.gasseri, L.acidophilus) Lactobacilli strains which are predominant in Europe, USA & Canada. Group III (L casei, L.paracasei, L.rhamnosus) were present in only 6% of women in Delhi. It was further found out that all strains of even the same species are not high secretors of lactic acid on the basis of their ability to make and secrete large amounts of lactic acid [12]. On the basis of their ability to make large quantities of lactic acid, positivity for making H₂O₂, high hydrophobicity and presence of arginine deiminase which prevents the formation of foul odour derivatives, we shortlisted three strains of Lactobacilli: L. salivarius TRF#30, L. fermentum TRF#36, and L. gasseri TRF#8. Figure 1 gives the species specific PCR profile of the three selected strains along with few others. (Table 2) summarizes the metabolic properties of the selected three strains of Probiotics. These meritorious Lactobacilli have been deposited in the Microbial Type Culture Collection and Gene Bank (MTCC), an International Depository Authority who have assigned the following numbers for these strains, L. gasseri (MTCC 5615), L. salivarius (MTCC5616) L. fermentum (MTCC 5617).

Table 2: Metabolic properties of the 3 meritorious strains of lactobacilli isolated from healthy vagina.

Figure 1: Species specific PCR products of the 10 species of Lactobacilli isolated from healthy vagina. This representative figure shows the profiles of 2 isolates of each of the 6 species and 1 isolate of the rest of 4 species as viewed on 2% agarose gel. S1 and S2, L. salivarius strains, 400 bp amplicon; R1 and R2, L. reuteri strains, 300 bp amplicon, P1 and P2, L. plantarum strains, 250 bp amplicon; F1 and F2, L. fermentum strains, 200 bp amplicon; G1 and G2, L. gasseri strains, 350bp product; C1 and C2, L. crispatus strains, 500bp PCR amplicon; J1, L. jensenii, 700 bp amplicon; A1, L. acidophilus, 200 bp PCR amplicon; PC, L. paracasei, 300 bp amplicon; Rh1 L. rhamnosus, 100 bp amplicon [9].

After individually testing these bacilli for their merit for restoration of healthy vagina, these three strains namely, L. salivarius TRF#30, L. fermentum TRF#36 and L. gasseri TRF#8 (hereafter denoted as Provag- Health) were selected for Phase II clinical trials.

After obtaining permission of The Drugs Controller General of India and approval of the Institutional Ethics Committees of the All India Institute of Medical Sciences (AIIMS) and Sir Ganga Ram Hospital New Delhi, 80 women suffering from recurring episodes of Vaginosis were enrolled for Phase II trials.

The criteria for enrolment of subjects were:

Enrollment criteria

(a) Women between age groups of 18-45 years with regular cycles.

(b) Clinical history of chronic and/ or recurring episodes of abnormal vaginal discharge. On pelvic examination, they are seen to have either excessive or unhealthy vaginal discharge.

(c) Willing to participate in the study after having been explained the nature of the study.

(d) Willing to return for follow up after 7 days of completion of treatment and thereafter on fortnightly basis for 2 months.

(e) Women enrolled should have telephone for recording by phone the discomfort, if any, after insertion of the capsules in the vagina.

Exclusion criteria

(a) Known case of diabetes mellitus.

(b) Suspected/confirmed pregnancy.

(c) Suspected or known malignancy of reproductive tract.

(d) Less than 6 weeks since last delivery or abortion since last delivery or abortion.

(e) H/O severe allergic reaction.

(f) Abnormalities of vaginal anatomy which will interfere with placement of drug.

(g) Known systemic diseases immune-compromised states.

(i) Adnexal mass or tenderness.

(j) Having received a course of antibiotic therapy, less than 14 days prior to enrollment.

(k) Dysfunctional uterine bleeding.

(l) Vaginal polyp.

The women enrolled were clinically examined and considered as suffering from Vaginosis on basis of thin white homogeneous abnormal vaginal discharge, presence of Clue cells indicative of microorganisms on epithelial cells of the vagina, pH of vaginal fluid above 5.0 and release of fishy odour on addition of 10% KOH (Whiff ’s test).

They were divided into four groups of 20 each. Twenty women in Group I was given the Pro-vag-Health Probiotics capsules. They were asked to take one capsule containing 3 × 10⁹ lyophilized stabilized lactobacilli every night for seven days. Group II women were given BASANT capsules 250 mg, one capsule to be taken every night for seven days. Group III of 20 women were given a Combination of Provag- Health and BASANT with the instruction to use one capsule of each every night for seven days. Group IV of 20 women were given placebo empty capsules to gauge the extent of spontaneous regression of Vaginosis over the observation time. The trials were randomized and women were distributed in these four groups with the help of a Statistician.

The efficacy of treatment with Polyherbal microbicide BASANT, Pro-vag-Health Probiotics and the Combination of the two hereafter named NAUROZ were gauged by:

(a) Absence of abnormal vaginal discharge.

(b) Absence of clue cells.

(c) Presence of lactobacilli.

(d) Absence of fishy odour.

(e) PH returning to below 5.0.

Figure 2 represents the typical findings of an effective treatment.

Figure 2: The Criteria employed to gauge the curative effect of treatment [11].

Pro-vag-Health Probiotics used by Group I women cured Vaginosis in 13 out of 20 women. BASANT was effective in 14 out of 20 women. Table 3 gives the findings in women treated effectively by BASANT. Table 4 represents a case, where Pro-vag-Health alone was unable to regress Vaginosis. Impressively the combination of the two, NAUROZ was effective in nearly every woman, 19 out of 20 women. Table 5 is a representative record of a patient using NAUROZ. Women were fully satisfied with the treatment. The subjects stopped coming to the clinic for follow- up even before completion of 2 months telling us on telephone that they do not feel the necessity of coming to us when they have no problem. In the fourth group of women, given Placebo capsules, only one woman out of 20 showed amelioration of her symptoms, while 19 others continued to suffer over the observation period [13]. Table 6 summarizes the overall findings of the trials. None of the women complained of any side effects.

Table 3: Representative record of the patient BV-A-107, who experienced regression of Vaginosis by intravaginal use of BASANT.

Table 4: Representative record of the Patient BV-A-100,who did not respond to the intake of (Pro-vag-Health) Probiotics.

Table 5: Representative record of the patient BV-A-127/II B, who experienced regression of Vaginosis by intravaginal use of BASANT+Pro-vag-Health.

Table 6: Summary of Results of treatment given to the four groups of women suffering from recurrent Vaginosis.

These trials lay the basis of treating Vaginosis by a Combination of the Polyherbal microbicide BASANT along with 3 meritorious strains of Probiotics. These were highly acceptable, fully safe, low cost, natural components. Being given that these patients come to the Clinics laden with aerobic and anaerobic microorganisms causing the syndrome, the efficacy of BASANT as an alternate to the antibiotics for elimination of the infecting microorganisms is noteworthy. Lactobacilli are present in the healthy vagina and perform the important task of bringing down the pH. Pro-vag-Health Probiotics are of human origin meritorious strains of Lactobacilli resident in healthy vagina. Their use recolonizes vagina with Lactobacilli to restore the acidic pH, hence these form part of the treatment in the Combination NAUROZ.

Before NAUROZ can be made available to the public, it is necessary to conduct Phase III trials which we plan to conduct in 120 women employing Metronidazole as comparator. Application has been filed to the Drugs Controller General of India for permission to conduct these trials. Meanwhile an established company HLL Life care (currently making Condoms) will make and supply BASANT capsules. The three meritorious strains of Lactobacilli, grown on a large scale, lyophilized along with stabilizers, will be supplied by M/s Microbax, Hyderabad.

We acknowledge with thanks the Research grants from the Indian Council of Medical Research (ICMR) and the Department of Biotechnology (DBT), Govt. of India for development of both BASANT & Probiotics. DBT also funded the Phase II clinical trial.