A comparative study of the therapeutic effects of the Zataria multiflora vaginal cream and metronidazole vaginal gel on bacterial vaginosis.

Abstract

Bacterial vaginosis (BV) is one of most prevalent complications among reproductive-aged women. Metromidazole prescription, which is considered as the first-line treatment of BV, is usually followed by a few side effects. Besides, there is a growing tendency toward herbal medicines for the treatment of vaginitis. Antibacterial and antifungal effects of Zataria multiflora (Z. multiflora) have been demonstrated in vitro and in vivo. This study aimed to compare the therapeutic effects of the Z. multiflora vaginal cream and metronidazole vaginal gel on bacterial vaginosis.

This was a randomized clinical trial on 90 married women aged 18-40 affected by BV who attended for treatment to the gynecology clinic of Shabih-Khani Hospital. They randomly divided to two groups of 45 participants. Diagnostic criteria were Amsel's criteria and Gram stain. Z. multiflora vaginal cream or metronidazole vaginal gel for five-night usage were prescribed to each group, and after 2-7 days therapeutic effects on participants' complications and their Amsel criteria were assessed. Data analysis was performed by McNemar and Fisher exact tests.

Patients' complication and their Amsel's criteria were significantly decreased after treatment with Z. multiflora or metronidazole (p < 0.05). Relative risk for unresponsiveness to treatment with Z. multiflora, to unresponsiveness to metronidazole was 1.5, which was not significant. Therapeutic effects of Z. multiflora vaginal cream are similar to metronidazole vaginal gel on BV. Therefore it could be an appropriate choice to BV treatment for those interested in herbal medicines or those affected by side effects of metronidazole.

[C] 2008 Elsevier GmbH. All rights reserved.

Keywords: Bacterial vaginosis; Zataria multiflora; Metronidazole

Introduction

Bacterial vaginosis (BV) results from an imbalance of the normal vaginal flora, with an overgrowth of anaerobic bacteria and a reduction in lactobacillary flora (Weir, 2004). Its prevalence was reported to be 27%, with 28% in sexually experienced and 18% in non-sexually experienced women (Yen et al., 2003). BV is associated with adverse pregnancy outcomes, upper genital tract infections such as pelvic inflammatory disease, endometritis, post-gynecologic-surgery infections, cervicitis, urinary tract infections, cervical intraepithelial neoplasia, and increased risk of sexual acquisition of human immunodeficiency virus infection (Cauci et al., 2002). Studies have shown that spontaneous abortion, preterm labor, premature birth, preterm rupture of the membranes, amniotic fluid infection, postpartum endometritis, and post-cesarean wound infections are increased because of infection with BV during pregnancy (McGregor and French, 2000). It is the most common vaginal infection among women of child-bearing age and in up to 25% of women attending gynecologic clinics (Sweet, 2000). Although metronidazole has been known as an effective management of BV, its efficacy must be weighed against its possible side effects (Pheifer et al., 1978). The most frequently reported side effects of metronidazole are vaginal discharge, symptomatic candidiasis, and vulvovaginal irritation; following oral metronidazole therapy, the side effects are gastrointestinal disorders, nausea, and metallic taste (Wain, 1998). The other option of management is clindamycin, which may cause dry skin, redness or irritation, peeling, oiliness, and itching or burning (Medlineplus, Clindamycine, 2007). The emergence of clindamycin-resistant anaerobic gram-negative rods following therapy is of concern (Beigi et al., 2004; Austin et al., 2005).

The use of herbal therapies is on the rise (Klepser and Klepser, 1999) and more than 80% of people in developing countries are using traditional medicines including herbal medicine (WHO, 2002). These medicines can be used safely if they are prescribed by educated health personnel with appropriate dose and special caution in pregnancy (Medlineplus, Herbal Medicine, 2008). The most commonly used alternative therapies for yeast vaginitis and BV are Lactobacillus recolonization by yogurt or capsules, boric acid douching, tea tree oil, and garlic (Kessel et al., 2003).

Thyme is a herbal remedy with antibacterial properties including gram-positive and -negative bacteria (Agnihotri and Vaidya, 1996). Its extract has been used to treat gastrointestinal disturbances, respiratory disorders, coughs due to colds, bronchitis and pertussis, laryngitis and tonsillitis, minor wounds, the common cold, disorders of the oral cavity, and as an antibacterial agent in oral hygiene (WHO, 1999).

Zataria multiflora is a thyme-like plant that grows wild in central and southern Iran, Afghanistan, and Pakistan. It is a member of the Labiatae family to which mint, rosemary, and several other medically useful plants also belong. The generic name derived from the Arabisc word "Za, atar" means Thyme. It can be recognized by the orbicular, densely gland-dotted, ovate leaves and the densely white hairy round buds in the leaf axils. It is an aromatic shrub with up to 60-89 cm in height. Mature branches are woody and leafless, whereas young branches are white with a dense glandular, spreading, pilose indumentums. Leaves (5-10 x 5-10 mm) are orbicular ovate to orbicular. Flowering stems are usually unbranched or sometimes have short lateral branches. Flowers are sub-sessile, very small, and often male sterile (Saleem, 2000). In Iran, Z. multiflora, called Shirazian thyme, is used as a traditional remedy for its antiseptic, analgesic, and carminative properties. It is also successfully used in the treatment of recurrent aphthous stomatitis (Jafari et al., 2003; Mansouri et al., 2002) and denture stomatitis (Amanlou et al., 2006).

The composition of the essential oil of Z. multiflora Boiss was studied by gas liquid chromatography (GLC), column chromatography (CC), NMR, and GLC/MS (Shafiee and Javidnia, 1997). The main constituents of the dry Z. multiflora are carvacrol (61.3%) and thymol (25.1%), while the main constituents of the fresh plant are thymol (48.4%), carvacrol (12.6%), P-cymene (13.5%), linalool (5.2%), and [gamma]-terpinene (3.9%) (Shafiee and Javidnia, 1997; Saleem et al., 2000, 2004). The essential oils reported from Z. multiflora are tricyclene, [alpha]-pinene, [beta]-pinene, camphene, [alpha]-terpineol, [rho]-cymene, myrecene, 1,8-cineol, [gamma]-terpinene, terpinolene, terpinen-4-ol, [alpha]-thujene, carvone, cavacrol, thymol, thymol acetate, methyl carvacrol, carvacrol acetate, aromadendrene, valencene, 9-aristolen-1-[alpha]ol, and guaiol, [alpha]-himachalene (Saleem, 2000).

Its essential oil was found to contain a significant amount of thymol and carvacrol, both of which are antimicrobial and antifungal (Shafiee and Javidnia, 1997; Saleem et al., 2004). The essential oil of Z. multiflora was also effective in impeding trichomonas in in vitro condition (Azadbakht et al., 2003). It has also demonstrated that Z. multiflora vaginal cream was as effective as clotrimazole vaginal cream in the treatment of Candida vaginitis (Islami et al., 2004).

This study aimed to compare the therapeutic effects of Z. multiflora (Loucorex[R]) vaginal cream with metronidazole vaginal gel on bacterial vaginosis, which seemed to be important with regard to the high prevalence of bacterial vaginosis and the general tendency towards herbal medicine.

Material and methods

This was a randomized clinical trial study, to compare the therapeutic effects of Z. multiflora vaginal cream (Loucorex[R]) with metronidazole vaginal gel on bacterial vaginosis. Ninety married women, affected by bacterial vaginosis, aged 18-40, participated in the study. They were randomly divided into two groups of 45 participants. The study was performed in the gynecology clinic of Shabih-Khani Hospital related to Kashan Medical Sciences University. These women were not pregnant; not breast feeding; not taking alcohol or were anti-coagulant, or were immunosuppressive- or IUD-users; and had no medical recognized disease. They were the only partner of their husbands. They were not using vaginal drugs during the last 14 days. They had no candidia or trichmonas vaginitis, or cervicitis based on direct smears. Written consent was taken from each participant before the commencement of the study. Ethical committee of Shahid Beheshti Medical University confirms the study.

Loucorex[R] was produced by Barij Essence Company (serial no. 1632). It was prepared as a cream in a 50 g tube, containing 0.315-0.385 mg thymol of Z. multiflora. Metronidazole was made by Pars Darou Company, as in a 0.75%g vaginal gel (serial no. 2625).

After history taking, the women were vaginally observed and examined using a speculum, to find out any signs of vaginitis and abnormal discharge. Samples of vaginal secretion specimen were collected using a sterile swab from the upper-lateral side of the vaginal wall. It was applied onto three slides. The first specimen was added by normal saline to observe the possible presence of clue cells and trichomonas vaginalis. The second specimen was added by one drop of 10% KOH solution to carry out the Whiff test and to assess the possible presence of candidia microscopically (x 40 magnification, USA Leica Company). The third specimen was dried out at room temperature and was fixed by methanol to be used for Gram stain and diagnosis of bacterial vaginosis by Nugent scoring. The sample pH was assessed using pH-meter paper (Nagel-Germany-Machery Co.). All the laboratory procedures were performed in the laboratory of Shabih-Khani Hospital. Women were excluded if there were trichomonas or candidia in their samples.

Criteria for diagnosis of BV were based on the presence of three Amsel's criteria of the following four criteria: a vaginal pH higher than 4.5; the presence of clue (vaginal epithelial) cells in the vaginal fluid; a thin, grey or white homogenous discharge; or a positive KOH "whiff" test (the release of an amine [fishy] odor upon the addition of 10% potassium hydroxide to the vaginal fluid). Also, Gram stain diagnosis of bacterial vaginosis was based on score 7-10 by Nugent criteria.

The treatment group of Z. multiflora used an application of the cream nightly for five nights. The treatment group of metronidazole used the gel vaginally for five nights. Some notes for personal health were also explained in a piece of paper for patients. A form for self-report was also given to all participants to assess their correct use of the medicines. The women were told to fill up the forms nightly, after the gel or the cream application. They were also asked to attend clinic 2-7 days following the treatment period. They were asked about drug complications. In this follow-up visit, whole diagnosis procedures were repeated again and successful treatment was demonstrated by the presence of one or no Amsel's criteria, and negative result of Gram stain.

Data were analyzed using SPSS and using tests such as Student's T test, Chi Square, Fisher exact test, and McNemar. McNemar was used to compare signs of bacterial vaginosis and Amsel's criteria, before and after treatment, and Fisher's exact test was also used to compare results following treatment between groups. The confidence interval for the tests was 95%.

Results

Ninety women affected by bacterial vaginosis were divided into two groups of 45. One woman from the metronidazole treatment group was excluded from the study because of incompatibility of Gram stain with Amsel's criteria. Two women from the Z. multiflora group were also excluded from the study because they did not attend the follow-up visits. Therefore full acceptance of the treatment was related to 88 women out of the 90 (97.8%) participants.

Treatment groups had no significant differences regarding their demographic and reproductive characteristics as well as their age, weight, years after marriage, education, age of first pregnancy, and number of pregnancy, abortion, or caesarian section (Student's T test, p > 0.05, Table l).Two groups were not significantly different regarding their occupation (Fisher's exact test, p > 0.05). There were also no significant differences between two groups regarding their contraceptive method and health condition (Fisher's exact test, p > 0.05, Table 2). The two groups were also not significantly different regarding the history of menstrual disorders or using wet underwear (Fisher's exact test, p > 0.05).

Table 1. Demographic and fertility characteristics of women with
bacterial in two treatment groups of Zataria multiflora and
metronidazole in Shabih-Khani Hospital of Kashan Medical University

      Characteristics                         Groups

                                 Z. multiflora      Metronidazole
                               (mean [+ or -] SD)  (mean[+ or -] SD)

Age(years)                    32.23 [+ or -] 5.82   32.7 [+ or -] 6.51
Weight (kg)                   66.35 [+ or -] 9.82     68 [+ or -] 10.37
Education (years)              9.05 [+ or -] 4.37   9.43 [+ or -] 4.46
Education of husband (years)  10.09 [+ or -] 3.75   9.52 [+ or -] 3.61
Matrial duration (years)      12.02 [+ or -] 5.95  12.30 [+ or -] 6.45
First pregnancy age (years)   19.77 [+ or -] 4.59  19.32 [+ or -] 2.88
Number of pregnancies          2.07 [+ or -] 1.12   2.25 [+ or -] 1.45
Number of caesarian sections   0.35 [+ or -] 0.65   0.36 [+ or -] 0.57
Number of abortions            0.12 [+ or -] 0.32   0.23 [+ or -] 0.52

Table 2. Contraceptive use and health condition of women with bacterial
vaginosis in two treatment groups of Zataria multiflora and
metronidazole Shabih-Khani Hospital of Kashan Medical University

Contraceptive and                          Groups
health condition

                           Zataria multiflora    Metronidazole no. (%)
                                 no. (%)

Contraceptive
  Combined oral                  4 (9.3)                4 (9.3)

Contraceptives
  DMPA                           1 (2.3)                0 (0)
  Condom                         4 (9.3)                3 (6.8)
  Withdrawal                    26 (60.5)              28 (63.8)
  Tubectomey                     3 (7)                  3 (6.8)
  Vasectomy                      1 (2.3)                1 (2.3)
  Candom and withdrawal          4 (9.3)                5 (11.4)

General health
  Pool-Swimming                  8 (18.6)              10 (22.7)
  Public bath use                1 (2.3)                3 (6.8)
  None                          34 (79.1)              31 (70.5)

Menstrual health
  Pad-use                       35 (81.4)              37 (84.1)
  Mattress-use                   5 (11.6)               3 (6.8)
  Cotton-use                     2 (4.7)                1 (2.3)

Total
  No. (%)                       43 (100)               44 (100)

The complications were significantly decreased in both groups of women. Except for itching, signs and symptoms were significantly decreased after the application of Z. multiflora cream (McNemar test, p < 0.05). The frequency of itching was decreased from 16.3 % to 4.7%, but this decrease was not significant. All signs and symptoms were significantly decreased after Metronidazole use (McNemar's test, p < 0.01). There were no significant differences between the two groups regarding the frequency of complications (Fisher's exact test, p < 0.01). Therefore, both treatment groups were effective in relieving patient's complications (Fig. 1).

[FIGURE 1 OMITTED]

There were significant differences between Amsel's criteria before and after Z. multiflora use (McNemar's test, p< 0.01). There were also significant differences between Amsel's criteria before and after metronidazole use (McNemar's test, p < 0.01). After treatment, Amsel's criteria were not significantly different between the two groups (Fisher's exact test p < 0.001). Therefore, both treatments had similar effects in improving Amsel's criteria after treatment (Fig. 2).

[FIGURE 2 OMITTED]

The relative risk for the failure of Z. multiflora to the failure of metronidazole in the treatment of BV was 1.53 (CI: 95%, 0.27-8.97). The relative risk was not significant and therefore both treatments have similar effects in the treatment of bacterial vaginosis (Table 3).

Table 3. Responses to the treatments of women with bacterial vaginosis
in two treatment groups of Z. multiflora and metronidazol

Treatment                          Groups

             Zataria multiflora no. (%)    Metronidazole no. (%)

Success              40 (93)                    42 (95.5)
Failure               3 (7)                      2 (4.5)
Total                43 (100)                   44 (100)

The complications of two treatment methods were assessed and demonstrated that Z. multiflora causes vaginal burning in 14% of users, although it does not cause metallic taste or vertigo (Table 4).

Table 4. Side effects of Zataria multiflora vaginal cream and
metronidazole vaginal gel in women with bacterial vaginosis

Treatment        Groups

                   Zataria multiflora no. (%)    Metronidazole no. (%)

Burning vagina               6 (14)                     2 (4.5)
Vaginal dryness              1 (2.3)                    1 (2.3)
Vertigo                      0 (0)                      2 (6.8)
Nausea                       1 (2.3)                    2 (4.5)
Metallic taste               0 (0)                      2 (4.5)
Total                       43 (100)                   44 (100)

Discussion

For the first time, this study demonstrated similar treatment effects of Z. multiflora cream and metronidazole gel on bacterial vaginosis. Similar treatment effects of the essence of Z. multiflora and clotrimazole cream on improving signs and symptoms of Candida vaginitis were demonstrated in previous studies (Islami et al., 2004 Fouladi, 2003). Z. multiflora is a thyme-like plant. Thyme is demonstrated to be as effective as clotrimazole in destroying the pathogen of Trichomonas vaginalis in vitro (Azadbakht et al., 2003). Thyme is one of World Health Organization-selected herbs with antibacterial and antifungal effects (Agnihotri and Vaidya, 1996; Cowan, 1999). Essential oil of thyme was demonstrated to have antifungal and anti-gram-positive and anti-gram-negative bacterial effects (Lopez et al., 2007) such as some Bacillus species (Ozcan et al., 2006) Staphylococcus aureus, Bacillus Subtilis, Escherichia coli (Fan and Chen, 2001) and Bacillus cerus (Akhound-Zadeh Basti et al., 2005). The antibacterial effect of the herb is related to phenolic components of thymol and carvacrol (Jafari et al., 2003). Carvacrol and thymol can alternate membrane fatty acid constituents of microbial cells (DiPasqua et al., 2006; Kelemba and Kunicka, 2003).

This study demonstrated that Z. multiflora vaginal cream has similar effects on improving the signs and symptoms of bacterial vaginosis and Amsel's criteria. The frequency of women with vaginal discharge, malodor, and itching was similar to a previous study (Gutman et al., 2005). A comparison between effects of Z. multiflora cream and clotrimazole cream for Candidia vaginalis demonstrated that Z. multiflora can reduce the frequency of vaginal discharge, itching, urine burning, and dyparonia, which was similar to the results of this study (Islami et al., 2004; Fouladi, 2003). The Z. multiflora vaginal cream was not effective in relieving itching in our study. However, there were only a few cases of itching before treatment that made statistical analysis impossible. So it cannot be concluded that Z. multiflora vaginal cream is not effective in improving the itching problem.

For the first time, this study showed that Z. Multiflora vaginal cream is effective in reliving bacterial vaginosis based on Amsel's criteria. The frequency of Amsel's criteria in BV patients of this study was similar to the frequency of other studies (Baloglu, 2003).

Metallic taste and vertigo was not caused by metronidazole; however, the frequency of nausea and vaginal dryness following the application of Z. multiflora was similar to after metronidazole use and burning vagina was even more frequent. Therefore, although Z. multiflora is usually reported without complication, thymol causes skin and mucosal irritation, and although there is no evidence for its teratogenic effects during pregnancy, its safety is not proved and its use is prohibited during pregnancy and breast feeding (Agnihotri and Vaidya, 1996).

One subject of the study in the metronidazole treatment group was excluded from the study because of incompatibility of Gram stain with Amsel's criteria. Two subjects were also excluded from the Z. Multiflora group because of their non-attendance in their follow-up visits. Therefore, accept abilities of the two treatment methods were 96% and 100% in the Z. multiflora and metronidazole groups, respectively.

Relative risk of failure in treatment was not significant, which means Z. Multiflora vaginal cream is as effective as metronidazole in the treatment of bacterial vaginosis. Bacterial vaginosis is the most prevalent cause of women attending gynecology clinics and besides it may cause critical complications. Therefore its treatment is important and metronidazole is the first treatment of choice and the most effective method of treatment of this condition. But metronidazole can cause a few side effects. Therefore, introducing a few alternative medicines may be useful. Essential oil of Z. multiflora is very effective on nonaerobic bacteria in vitro. Since Lactobacillus sp., which is a useful flora of vagina, is resistant to Z. multiflora, metronidazole is an appropriate alternative medicine for bacterial vaginosis.

In conclusion, herbal medicine and their healthy production can make a wider range of treatment options for patients and practitioners. Herbal medicines have limited complications and can be appropriate alternatives for chemical medicines. With regard to the long history of herbal medicine in Iran, women's acceptability of these medicines is high and standard production of these drugs can make their prescription by health personnel easier. Obstetrician and midwives can offer Z. multiflora as an appropriate alternative medicine for women who like to use herbal medicines, or who suffer metronidazole side effects in the treatment of bacterial vaginosis.

Acknowledgement

We appreciate the research department of Shahid Beheshti Medical University, which found the study and Gynecology clinic as well as laboratory personnel of Shabih-Khany Hospital for their friendly cooperation with the researchers of the study.

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M. Simbar (a), *, Z. Azarbad (b), F. Mojab (c), H.Alavi Majd (d)

(a) School of Midwifery, Shahid Beheshti Medical Science University, Tehran, Iran

(b) Deputy of Research, Kashan Medical Science University, Kashan, Iran

(c) School of Pharmacy and Pharmaceutical Sciences Research Center (PSRC), Shahid Beheshti Medical Science University, Tehran, Iran

(d) School of Paramedical, Shahid Beheshti Medical Science University, Tehran, Iran

* Corresponding author. Tel.: + 98 21 8888 3133;

fax: + 98 21 4400 8378.

E-mail addresses: [e-mail], [e-mail] (M. Simbar).

0944-7113/$-see front matter [C] 2008 Elsevier GmbH. All rights reserved.

doi: 10.1016/j.phymed.2008.08.004