Comparison of the effect of propolis vaginal gel and metronidazole oral tablet on wet smear test results in women suffering from trichomoniasis

doi:10.21203/rs.3.rs-2084927/v1

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Comparison of the effect of propolis vaginal gel and metronidazole oral tablet on wet smear test results in women suffering from trichomoniasis

https://doi.org/10.21203/rs.3.rs-2084927/v1

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Background and objective: Trichomoniasis is the most common non-viral sexually transmitted infection. One of the methods for diagnosing trichomoniasis is a wet smear test. Propolis has anti-parasitic properties are due to its components, namely tannins, flavonoids, and volatile oils. The present study was performed to compare the effect of a vaginal gel containing propolis extract with that of a metronidazole oral tablet on the wet smear test results in women suffering from trichomoniasis.

Material and method: The present clinical trial was performed on 102 married non-pregnant women with trichomoniasis, who were referred to obstetrics and gynecology clinics in Isfahan from September 15 to January 20, 2020. The patients were assigned into two groups of intervention and control randomly. Both groups underwent treatment for 7 days. Wet smear was taken from the patients three times (i.e. initial visit, first follow-up, and second follow-up) and the samples were observed under Carl Zeiss single-lens microscope. Data were analyzed using SPSS (version 20) at a significance level of 0.05.

Results: Two groups did not differ significantly in terms of baseline characteristics. In the first post-treatment follow-up, the chance of a negative wet smear test result was 4.41 times greater in the intervention group than in the control group. Given the control of the baseline characteristics, this chance was 5.8 times greater. In addition, it was found that the chance of a negative wet smear test result in the second follow-up was affected by the wet smear test result in the first follow-up.

Conclusion: Based on the results of this study, it can be concluded that vaginal gel containing 7.5% propolis extract was more efficient than standard treatment in eradicating infection and preventing the recurrence of infection.

Propolis

vaginalis

Trichomoniasis

Wet smear

Iran

Trichomoniasis is the most common non-viral of the worldwide spread of sexually transmitted diseases (1–3), the prevalence of trichomoniasis in the world is more than that of gonorrhea, syphilis, and chlamydia infection. About 276.4 million new cases of trichomoniasis occur each year among adults aged 15–15 years. The prevalence of this infection in the United States was 25% in women referred to sexually transmitted disease clinics, and it was reported as 3.2% in some Islamic countries, such as Turkey, Libya, and Jordan (4–7). According to various studies, the prevalence of it in Iran was estimated to be about 1–37%. Its prevalence was reported to be 1.1% in Karaj, 3.3% in Zanjan, 2.1% in Hamedan, and 24.4% in Isfahan (8–11).

Trichomonas is clinically important because, in addition to the unpleasant and annoying symptoms of vaginitis, is associated with several serious conditions like atypical pelvic inflammatory disease (PID), infertility, a high likelihood of HIV acquisition and transmission infection, human papillomavirus, cervical cancer, and cervix malignancies, the transmission of other sexually transmitted infections, and adverse pregnancy outcomes (1, 2, 4, 5, 12, 13)

Sexually transmitted infections (STIs) in low- and middle-income countries are predominantly managed by syndromic management, but they can manage in men and women based on laboratory tests. Trichomonas vaginalis in women can be isolated from vaginal secretions, prostate, urethra, semen, and urine. The usual method for diagnosing trichomonas is a microscopic examination called wet smear (1, 13–16). Vaginal discharge can be checked by preparing a wet smear sample. For this purpose, the vaginal sample is placed in 0.5 ccs of saline in a tube, then it is transferred to a slide, a cover slip is placed on the slide, and then a microscopic examination is done. Some physicians prefer to mix a sample of vaginal discharge with saline directly on the slide. In this method, the organism remains motile for 10–20 minutes after collecting the sample. The sample should be studied as soon as possible because the sensitivity of the test decreases over time so a sensitivity reduction of more than 20% has been reported after one hour of sampling. The advantages of this method are its ease, low cost, and speed of detection. The sensitivity of this method in comparison with parasite culture in Diamond’s medium is less and about 45 to 60%. About 60 to 70% of culture-positive samples can be detected by wet smear test (2, 7, 14, 15, 17). her diagnostic methods include culture nucleic acid amplification test (NAAT), rapid antigen or nucleic acid probe test, quantitative real-time, and Pap smear (1, 7). NAAT is the most sensitive and specific test for trichomonas and it is considered the gold standard for trichomonas diagnosis (1, 14). As in the present study, the conventional method of diagnosing trichomonas vaginalis; the wet smear test, was used as an evaluation method and as a criterion for recruiting research units in both groups due to its speed, ease of sampling, and acceptable sensitivity and specificity.

Metronidazole and tinidazole are the drugs of choice for the treatment of trichomoniasis. Metronidazole has been the only approved drug for the treatment of this infectious agent since 1961. Treatment with metronidazole has always faced two challenges treatment failure and drug resistance.

The CDC has estimated that the resistance of isolated parasites is 2–5%. Following drug resistance, we will face an increase in dose and subsequently an increase in drug side effects. These side effects include headache (12%), nausea (10–12%), metallic taste in the mouth (9%), genital itching (5%), dizziness (4%), diarrhea (4%), and dry mouth (2%). (1, 3, 6, 7). The growing trend of drug resistance and numerous side effects has led researchers to seek drugs with fewer side effects, lower prices, and more accessibility as well as drugs from natural sources, such as medicinal plants (1, 7). One of these compounds is propolis. Propolis is a resinous-like substance collected by bees from plants, flower buds, and gums (22, 24). Generally, propolis consists of 50% plant gum (resin), 30% wax, 10% essential and aromatic oils, 5% pollen, and 5% other organic and inorganic substances (18, 19). The main compounds of propolis include phenolic compounds (phenolic acids), flavonoids, terpenoids, aromatic aldehydes, steroids, alcohols, fatty acids, hydrocarbons, sugars, and minerals. Flavonoids, terpenoids, and phenolic esters propolis are responsible for the propolis main effects (18–20). The antioxidant (21), immunomodulatory (22), antimicrobial, antifungal (23) and antiparasitic (24)properties of propolis have been investigated. In addition, the wound-healing properties of propolis have been proven (25).

Due to the high prevalence of trichomoniasis, complications induced by the disease, increased drug resistance, and side effects associated with antiparasitic drugs and given the proven antiparasitic effects of propolis, the present study was performed to compare the effect of a vaginal gel containing propolis extract with that of metronidazole oral tablet on the wet smear test results in women with trichomoniasis.

Study design

The present study was a clinical trial that included two groups and three phases. The study aimed was to compare the effect of a vaginal gel containing propolis extract and metronidazole tablets on wet smear test results. The participants included 102 married non-pregnant women with trichomoniasis, who were referred to obstetrics and gynecology clinics of Amin, Shahid Beheshti, and Al-Zahra in Isfahan from September 15 to January 20, 2020. This study was confirmed by the Ethics Committee of Isfahan University of Medical Sciences (ethics code: IR.MUI.REC.1398.001) and was registered at the Iranian Registry of Clinical Trials (IRCT20091219002889N10).

Participants

Among women referred to the clinics, those who met the inclusion criteria were selected. The inclusion criteria were as follows: provision of written consent, having Iranian nationality, being at reproductive age (18-45), married, and monogamous, having a positive wet smear test, and being clinically diagnosed with trichomoniasis. Patients who were unwilling to continue the treatment, pregnant or breastfeeding, in the menstrual cycle at the time of investigating clinical symptoms, did not refer for follow-up visits, did not use the prescribed drug regularly and according to the instructions (not taking the drug for two consecutive nights or three non-consecutive nights/days) as well as those who had a history of drug allergy or any other allergies, underwent trichomoniasis treatment in the past month, hormone therapy or immunosuppressive drugs, had a history of a known case disease or chronic disease (chronic diabetes, anemia, cardiovascular diseases, kidney or liver diseases, different types of cancers, and thalassemia) and history pelvic surgery, took other antibiotics or underwent other supportive therapies such as vaginal douching during the study, and had sex without a condom during the study were excluded. After providing explanations about the treatment methods to the patients and obtaining written informed consent from them, the study was initiated.

Sampling

Patients were selected through a convenience sampling method while considering the sample size estimated and inclusion criteria defined in this study. The patients were randomly divided into two groups intervention (consuming propolis vaginal gel) and control (taking metronidazole oral tablets) randomly. For assigning the patients into two groups of intervention and control, each patient was asked to choose one envelope out of 110 envelopes (it was written “control group” on 51 envelopes and “intervention group” on the other 51 envelopes). After 109 times, the sample size reached adequate (51 patients in each group). It should be noted that each envelope selected by the patients was returned and thus everyone had the same chance in choosing the envelope.

Intervention

The vaginal gel used in this study contained 7.5% of propolis extract. The gel was prepared by combining 90 g of propolis with 1110 g of cream base (produced by Daru Darman Salafchegan Company) at 40⁰ C and subsequently obtaining 1200 g of propolis compound 7.5%. It was then packed in 60 tubes of 20 g. The selected drug dose was based on studies done by Mousavi (15) and Mohammadizadeh et al (26). The drug was produced by the Pharmaceutical Company of Daru Darman Salafchegan and then provided to the researcher. Microscopic observation of vaginal wet smear was also performed by first inserting a speculum and then collecting a sample of cervical and posterior fornix mucosal secretions using a sterile swab. The swab was placed inside a test tube containing 0.5 ccs of physiological saline. Then a drop of the sample was placed on the slide and examined under a microscope with a magnification power of 20× and 40× for direct diagnosis of the parasite. All the patients received the same education on not using the vaginal douche, not having sex without a condom during the study, the amount of physical activity, and nutritional tips through face-to-face training and educational pamphlets. The patients in both groups were coded appropriately by the researcher. After completing the patients’ basic information, the researcher provided the vaginal gel with seven disposable applicators to the intervention group and 14 metronidazole 500 mg tablets, as a standard treatment for trichomoniasis, to the control group. The researcher asked the intervention group to put the gel containing propolis extract in the vagina every night before bed for a week. The patients in the intervention group were advised to lie on their backs for at least 30 minutes after using the gel and continue to use the gel for seven nights. The control group was also asked to take 500 mg metronidazole tablets twice daily every 12 hours for one week. The patients in both groups were asked not to take any other medications during the study. In both intervention and control groups, sexual partners were treated simultaneously by taking metronidazole 500mg tablets twice daily every 12 hours for one week. All the patients treated with metronidazole were advised to abstain from alcohol for up to 24 hours after discontinuation of treatment due to the possibility of a disulfiram-like reaction. in the end, all the patients who participated in the study were praised and thanked.

Follow-up

During the treatment period, the use of medication and health tips were reminded to the research units twice by phone calls made by the researcher. Immediately and two weeks after the intervention, the patients were asked to refer to a pre-determined clinic for clinical examination and completion of the form by the researcher. In case of no clinical improvement or worsening of the clinical condition in the first follow-up (immediately after the intervention) or second follow-up (two weeks after the intervention) in the intervention group, the standard treatment was started (i.e. metronidazole oral tablets). In addition, in case of non-response to treatment, the patient was referred to a gynecologist to receive a complete and standard treatment freely. In the event of the occurrence of any of the exclusion criteria that had already been warned to the research units, the patient was excluded from the study.

Study instruments

Data collection in this study was done through direct observation of the parasite by Carl Zeiss single-lens microscope made in Germany and completion of demographic information form (age, race, education, economic status, etc.), obstetrics and gynecology checklist, a questionnaire on clinical signs and symptoms of trichomoniasis, and a form detailing daily drug use. The content validity of the researcher-made questionnaire was confirmed by ten professors of the School of Midwifery and Nursing at Isfahan University of Medical Sciences.

Statistical analysis

Following data collection, data analysis was done using SPSS (version 20) and running statistical tests of independent sample t-test, chi-square test, Fisher's exact test, ANOVA, and logistic regression. α=0.05 was considered a significant level.

The current clinical trial consisted of three phases and was conducted on 102 non-pregnant married women suffering from trichomoniasis. Initially, 109 patients were recruited. During the sampling process, seven patients were excluded from the study, including four from the control group and three from the intervention group. In the control group, three patients were excluded due to loss of follow-ups and one was excluded due to non-intermittent and incorrect use of the drug. In the intervention group, one patient was excluded due to loss of follow-ups, one due to incorrect use of the drug, and one due to lack of treatment of sexual partner (Consort Diagram).

The results showed that the two groups under study are homogeneous and no statistically significant difference was observed in baseline characteristics. In other words, the results of the statistical tests confirm the random allocation of the studied samples into two test and control groups (Table 1).

Table 1

Comparison of basic underlying variables between intervention and control groups
Variable		Number (percent)
Variable		Intervention group	Control group
Education level	Secondary education or lower	((84.3 43	38 (74.5)
Education level	Undergraduate	8 (15.7)	13 (25.5)
Occupation	Housewife	38 (74.5)	39 (76.5)
Occupation	Employed	13 (25.5)	12 (23.5)
Husband’s occupation	Worker	5 (9.8)	3 (5.9)
	Employee	6 (11.8)	6 (11.8)
	Self-employed	40 (78.4)	42 (82.4)
Husband’s education level	Secondary education or lower	43 (84.3)	39 (76.5)
Husband’s education level	Undergraduate	8 (15.7)	12 (23.5)
History of infection	No	20 (39.2)	24 (47.1)
History of infection	Yes	31 (60.8)	27 (59.2)
Presence of infection symptoms in the sexual partner	No	44 (86.3)	40 (78.4)
	Pain	1 (2)	3 (5.9)
	Burning	5 (9.8)	5 (9.8)
	Pain and burning	1 (2)	3 (5.9)
Mean ± standard deviation
Age		7.31 ± 36.29	7.74 ± 35.67
Husband’s age		9.16 ± 42.18	9.44 ± 40.75
Number of pregnancies		1.54 ± 2.12	1.55 ± 2.16
Number of deliveries		1.35 ± 1.82	1.29 ± 1.75

Based on the results of Table 2, the chance of a negative wet smear test immediately after completing the treatment was 4.41 times greater in the intervention group than in the control group, indicating a statistically significant difference between groups (p = 00.1).

Table 2

Comparison of the difference in negative wet smear test immediately after completion of the treatment in the intervention and control groups
Study groups	test result in Wet smear		Odds ratio (OR)	95 %confidence interval OR		P-Value^*
	Positive Number(percent)	Negative Number(percent)		Minimum	Maximum
Intervention group	28 (54.9)	23 (45.1)	4.41	1.73	11.24	0.001
Control group	43 (84.3)	8 (15.7)
* Chi-Square

As depicted in Table 3, the odds ratio (OR) of the negative wet smear test immediately after completing the treatment was higher in the intervention group than in the control group in patients with normal delivery, history of all levels of the previous infection, lack of symptoms of infection in a sexual partner, and housekeeping job.

Table 3

Comparison of the difference in the negativity of wet smear test results immediately after the completion of the treatment in the intervention and control groups concerning underlying variables
Variables		Study groups		Wet smear test result		OR	95 %confidence interval OR				P-Value
				Positive Number(percent)	Negative Number(percent)		Minimum		Maximum
Type of delivery		No delivery	Intervention group	3 (42.9)	4 (57.1)	9.33	0.71	122.57		^*0.067
			Control group	7 (87.5)	1 (12.5)
		vaginal	Intervention group	20 (57.1)	15 (42.9)	5.75	1.45	22.78		^*0.008
			Control group	23 (88.5)	3 (11.5)
		Cesarean section	Intervention group	5 (55.6)	4 (44.4)	2.6	0.46	14.63		^*0.272
			Control group	13 (76.5)	4 (23.5)
History of infection	No	Intervention group		6 (45)	11 (55)	3.66	1.02	13.14		^*0.042
		Control group		18 (75)	6 (25)
	Yes	Intervention group		19 (61.3)	12 (38.7)	7.89	1.57	39.55		^*0.005
		Control group		25 (92.6)	2 (7.4)
The presence of a symptom of infection in the sexual partner	No	Intervention group		25 (56.8)	19 (43.2)	3.4	1.14	8.08		^**0.035
		Control group		32 (80)	8 (20)
	Yes	Intervention group		0	1 (100)	-	-	-		^**0.25
		Control group		3 (100)	0
	Burning	Intervention group		3 (60)	2 (40)	-	-	-		^**0.444
		Control group		5 (100)	0
	Pain and burning	Intervention group		0	1 (100)	-	-	-		^**0.25
		Control group		3 (100)	0
Occupation	Housewife	Intervention group		20 (52.6)	18 (47.4)	6.12	1.96	19.02		^*0.001
		Control group		34 (87.2)	5 (12.8)
	Employed	Intervention group		8 (61.5)	5 (38.5)	1.87	0.33	10.46		^*0.471
		Control group		9 (75)	3 (25)
* Chi-Square
** Fisher's Exact Test

Given the control of variables of occupation, the type of delivery, the history of infection, and the presence of symptoms in the sexual partner, the chance of a negative wet smear test immediately after treatment was 5.8 times greater in the intervention group than in the control group (Table 4).

Table 4

Comparison of the difference in the negativity of wet smear test result from two weeks after the completion of treatment in the intervention and control groups with respect to wet smear test result immediately after the completion of treatment
Variable	Study groups		Wet smear test result two weeks after the completion of treatment		OR	95 %confidence interval		P-Value^*
			Positive Number(percent)	Negative Number(percent)		Minimum	Maximum
Wet smear test result immediately after the treatment	Positive	Intervention group	14 (50)	14 (50)	1.52	0.58	3.99	0.465
		Control group	26 (60.5)	17 (39.5)
	Negative	Intervention group	(0)	23 (100)	-	-	-	0.258
		Control group	1 (12.5)	7 (78.5)
* Fisher's Exact Test

According to Table 5, the OR of the negative wet smear test two weeks after the completion of the treatment in the intervention group compared to the control group was affected by the result of the wet smear test immediately after the completion of the treatment. Based on our findings, there was no difference between the two groups in terms of the chance of having a negative test two weeks after completion of the treatment.

Table 5

Logistic regression results on the negativity of wet smear test result immediately after completion of the treatment in the intervention group compared to the control group, adjusted for occupation, type of delivery, history of infection, and the presence of symptoms in the sexual partner
Study groups	B	S.E	Wald	OR	95 %confidence interval		P-Value^*
Study groups	B	S.E	Wald	OR	Minimum	Maximum	P-Value^*
Control group	Reference				-
Intervention group	1.75	0.525	11.20	5.80	2.07	16.26	0.001
* Logistic Regression

In this study, a significant difference was detected between the intervention group and the control group in terms of wet smear test results. According to the results of this study, the chance of obtaining a negative result in the first follow-up was 4.41 times greater in the intervention group than in the control group. Considering the underlying variables in patients with a housekeeping job, vaginal delivery, all levels of the history of infection, and the lack of symptoms of infection in the sexual partner, the chance of a negative wet smear test result was higher in the intervention group than in the control group. Provided that these variables were adjusted, this chance in the first follow-up was 4.41 times greater in the intervention group than in the control group, indicating higher efficacy of gel-containing propolis over the standard treatment in the treatment of trichomoniasis. The wet smear test results in the second follow-up were affected by the result of this test in the first follow-up. In other words, the positivity or negativity of the test results in the second follow-up did not change significantly compared to that in the first follow-up. our findings indicated the consistency of wet smear test results in the first and second follow-ups in the intervention group, suggesting the greater efficacy of the vaginal gel containing propolis extract in comparison with metronidazole in preventing the recurrence of infection. Santa et al., (1992) investigated the effect of propolis powder in the form of a vaginal douche on acute cervicitis, trichomoniasis vaginalis, and Gardnerella vaginalis (27). In addition to clinical symptoms, vaginal discharge culture before treatment and one week after the end of treatment were studied. The results of this study showed that ten days after treatment, 100% of the patients who showed symptoms of acute cervicitis achieved complete recovery and at the same time their wet smear test result was negative, which was in line with the findings of this study. Abdali et al., (2015) compared the effect of 0.1% vaginal Zataria multiflora cream and oral metronidazole tablets in women of childbearing age suffering from trichomoniasis and Gardnerella vaginal infections (28). It should be noted that Zataria multiflora contains 69% phenolic components, saponins, caffeic acid, resin, tannin, resonates, and 2.6% volatile oil and is very similar to propolis in terms of components. The results of the wet smear test after treatment led to a significant difference between the intervention and control groups (p = 0.001), which was consistent with the results of Abdali et al.’ study. Dubouchet et al., (1996) compared the efficacy of clotrimazole vaginal tablets, oral metronidazole, and vaginal suppositories containing sulfanilamide, aminacrine hydrochloride, and allantoin in the treatment of symptomatic trichomoniasis (29). Positive wet smear test results were observed in 81.5% of patients whose culture test was also positive after treatment. However, negative culture test results were reported after treatment in about 80% of patients who received metronidazole at a dose of 2 g. While 11% of the topical clotrimazole group and 18.6% of the sulfanilamide-aminacarcin-allantoin group had negative test results. The strengths of the study were the novelty of the research using vaginal propolis extract for the first time in an interventional study, control of interfering factors, measurement of pain severity by one person (the main researcher), and homogeneity of the two groups in terms of quantitative and qualitative underlying variables. In addition, the patients were followed up until complete treatment if they did not improve. One of the limitations of the study was the lack of use of placebo in both groups to blind the research units and the researcher to the allocated treatment.

Considering the results of this study, it can be concluded that vaginal gel containing 7.5% propolis extract was more efficient than standard treatment in eradicating infection and preventing the recurrence of infection. Since the present study was the first one that used vaginal propolis in the treatment of trichomoniasis, further research investigating other gynecological diseases and the effectiveness of different doses of propolis is recommended.

Conflict of interest

None to declare.

Acknowledgments

The present study was part of a postgraduate thesis in midwifery presented by Shadi Taghavian (No 397670). This study was funded by the Research Deputy of Isfahan University of Medical Sciences. We are thankful to the staff working at Research Deputy and Department of Midwifery and Reproductive Health, Faculty of Nursing and Midwifery, Isfahan University of Medical Sciences, clinics of Amin, Shahid Ayatollah Beheshti, and Al-Zahra in Isfahan, and all the patients participated in the research.

Ethical Approval

This study was confirmed by the Ethics Committee of Isfahan University of Medical Sciences (ethics code: IR.MUI.REC.1398.001) and was registered at the Iranian Registry of Clinical Trials (IRCT20091219002889N10).

Authors' contributions

1- Taqavian Sh= Designed the study and wrote the main manuscript text.

2- Vliani M*= She is the responsible author and Designed the study.

3- Beiranvand R= statistics analysis and prepared figures 1-5.

4- Alian S= he prepared the drug that was used for the study and helped to design the study.

5- Yekdaneh A= She helped to prepare the drug that was used for the study and helped to design the study.

6- Tehrani GH= She helped with the intervention of the study.

7- Zafarbakhah A= She helped with the intervention of the study.

8- Khalili T= She helped with the intervention of the study.

Funding:

All of this study’s funding was received from the Isfahan University of medical sciences.

Availability of data and materials:

The SPSS file of data analyses and all of the questionnaires are available. We send this if you want.

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No competing interests reported.

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Comparison of the effect of propolis vaginal gel and metronidazole oral tablet on wet smear test results in women suffering from trichomoniasis

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