Trichomonas vaginalis is the causative agent of the most common curable sexually transmitted disease (STD) in the world.1 The annual incidence in the United States is estimated to be 5 million cases.2 Although the infection is associated with increased risk for HIV acquisition and adverse outcomes of pregnancy,3,4 control efforts for trichomoniasis have lagged behind those for other STDs. Trichomonas causes vaginitis in women5 and is either asymptomatic or causes symptomatic urethritis in men.6 The infection is treated with a single oral dose of metronidazole or tinidazole, currently the only licensed class of drugs available for this indication; however, both of these antimicrobials are associated with significant gastrointestinal adverse effects, and some individuals are unable to tolerate them because of these adverse effects.7
Neo-Penotran Forte (Embil Pharmaceuticals, Istanbul, Turkey) is a combination intravaginal product that contains 750 mg of metronidazole plus 200 mg of miconazole. A similar product containing 500 mg of metronidazole (Neo-Penotran; Embil Pharmaceuticals) has been studied for the treatment of vaginitis with promising results.8,9 We conducted a pilot study of 750 mg metronidazole/200 mg miconazole to determine the potential efficacy of this high-dose metronidazole intravaginal product for the treatment of vaginal trichomoniasis.
MATERIALS AND METHODS
Women 19 years or older attending the Jefferson County STD Clinic in Birmingham, Alabama, were recruited for this pilot study from December 2011 and July 2012. Women attending the STD clinic are routinely screened for trichomoniasis using wet prep microscopy of the vaginal fluid. Women diagnosed as having trichomoniasis were notified of the opportunity to participate in this study. The study was approved by the institutional review board at the University of Alabama at Birmingham and registered with ClinicalTrials.gov (NCT01361048). Women were excluded from the study if they were pregnant or nursing; were allergic to metronidazole or miconazole; had coinfection with yeast seen on wet prep; had a history of seizures or peripheral neuropathy; required lithium, warfarin, or disulfiram; or were expecting to have menses within 8 days of enrollment. At enrollment, women completed baseline questionnaires and a vaginal swab was obtained for inoculation of culture media for trichomonas. This randomized, dose-ranging pilot study was conducted in 2 phases consisting of 20 participants in each phase. In the first phase, participants were randomized to the vaginal suppository (metronidazole 750 mg/miconazole nitrate 200 mg) twice a day for 7 days versus oral metronidazole 2 g single dose, the latter administered as directly observed therapy. In the second phase, participants randomized to suppository used it once a day for 7 days. The randomization sequence was computer generated for each study phase using block randomization with blocks of sizes 4 and 6. Sealed envelopes with treatment assignment were opened after women completed the baseline questionnaire. Women were asked to abstain from alcohol ingestion during the treatment period and to abstain from unprotected sex during the course of the study. They were advised to inform their partner(s) of the need for therapy. Women were asked to return for reevaluation on days 12 to 15 and 30 to 35 of the study. At these visits, follow-up questionnaires were administered and vaginal swabs obtained for wet prep and culture. Clinical staff were not blinded to treatment assignment for this study. However, laboratory staff who interpreted the cultures for trichomonas were unaware of treatment assignment. Treatment failures were defined as persistence of trichomonas by wet prep and/or culture. Women who failed therapy were treated with 2 g of single-dose oral metronidazole and discontinued from the study.
T. vaginalis was isolated from the vaginal vault of study participants during a standardized physical examination and cultured in T. vaginalis InPouch media (Biomed Diagnostics, White City, OR). Axenic cultures were subsequently prepared in modified Diamond medium by subculture to remove contaminating human cellular material, yeast, and bacteria. T. vaginalis DNA was extracted using the Generations Kit (Qiagen, Valencia, CA). The concentration of DNA was determined by direct measurement with a Nanodrop (Wilmington, DE) spectrophotometer. Template DNA was diluted to a concentration of 5 ng/μL. Random amplification of polymorphic DNA (RAPD) was performed using 6 low-stringency polymerase chain reaction reactions, each containing one unique primer, 25 ng of template DNA, and an amplification “bead” containing the polymerase chain reaction buffer, dNTPs, and Taq polymerase (Illustra Ready-To-Go RAPD Analysis Kit; GE Healthcare Biosciences, Piscataway, NJ). Reaction parameters were as follows: 95°C × 5 minutes/(95°C × 1 minute; 36°C × 1 minute; 72°C × 2 minute) × 45 cycles/4°C hold. Amplicons were eletrophoresed in 2% agarose with tris acetate EDTA buffer prestained with ethidium bromide. Amplicon sizes were determined by comparison with a molecular-weight ladder (Hi-Lo DNA Marker, Minneapolis, MN) using Quantity One software (Bio-Rad Life Sciences, Hercules, CA).
Participant characteristics and laboratory results were compared according to treatment arm separately for each phase using Fisher exact tests for categorical data, exact Cochran-Armitage trend tests for ordinal data, and t tests for continuous data. Similarly, the cure rates at the first and second follow-up visit were compared according to arm using Fisher exact test for each phase. Cure was defined as a negative test result by both wet mount and culture. An overall efficacy response was similarly analyzed that carried forward the test result from the first follow-up visit when the result at the second follow-up visit was missing. Odds ratios and 95% confidence intervals were computed, and exact conditional methods were used when there were no treatment failures in a group. A combined analysis was also performed for the common odds ratio across phases, which ignored dosing in the suppository arm, using an exact Mantel-Haenszel χ2 test. For this pilot study, the sample size determination was based on practical considerations, not statistical calculations. Analyses were performed using SAS 9.3.
Participant flow is presented in Figure 1. Patient characteristics for each phase and treatment group are shown in Table1. There were no significant arm differences in either phase, except for age in phase 2 (mean, 28.3 vs. 36.9 years for suppository vs. oral; P = 0.031). For all enrolled women, 30 (75%) of 40 presented for evaluation of vaginal symptoms. A history of any STD was reported by 35 (87.5%) of 40 women, and 21 (52.5%) of 40 reported a history of trichomoniasis. There was no significant difference between treatment failures and cures in terms of reported interim sexual activity or partner treatment (data not shown). Table 2 shows the baseline laboratory results for all groups. There were no significant differences between treatment arms for either phase. Overall, the mean vaginal pH was 6.0, the Whiff test was positive in 33 (82.5%) of 40, clue cells were present in 18 (45%) of 40, and none of the women had concomitant yeast infections.
Cure rates for all groups are presented in Table 3. There were no significant differences in cure rates between Neo-Penotran vaginal suppositories and oral metronidazole in either phase. At follow-up visit 1, the cure rate was 90% versus 100% for the suppository (2×/d) versus oral medication arms in phase 1 (P = 1.00) and 88% versus 80% for the suppository (1×/d) versus oral medication arms in phase 2 (P = 1.00). At follow-up visit 2, the cure rate was 88% versus 88% for the suppository (2×/d) versus oral medication arms in phase 1 (P = 1.00) and 88% versus 88% for the suppository (1×/d) versus oral medication arms in phase 2 (P = 1.00). The overall efficacy across both follow-up visits was 80% versus 90% for the suppository (2×/d) versus oral medication arms in phase 1 (P = 1.00) and 78% versus 70% for the suppository (1×/d) versus oral medication arms in phase 2 (P = 1.00). The results were also nonsignificant when combining results across arm (P = 1.00).
There were 21 adverse events in 18 participants. All were mild, except for one moderate adverse event of suicidal ideation, which was also the only serious adverse event in the study. This occurred at the final visit and was felt to be unrelated to treatment. There were 10 adverse events that were related to study medication in 9 participants, all of which were mild. The most common was vaginal itch (3 events in 3 different participants), all in the first phase with 1 in the suppository arm and 2 in the oral medication arm. There was one gastrointestinal adverse event in the study (dysgeusia), which occurred in the second phase in the suppository arm and was deemed related to study medication.
To assess true treatment failure versus reinfection, we performed the RAPD molecular technique to create a “fingerprint” of T. vaginalis visit-specific isolates.10 Of the 8 participants with T. vaginalis detected at the follow-up visits, 6 had viable specimens for 2 visits, baseline and a follow-up. All RAPD patterns were discordant for all visit pairs. Percent discordance ranged from 58.6% to 93.5% (Fig. 2). RAPD patterns were also analyzed if banding patterns of the treatment failure visit could be “subtracted” from the initial visit as a check to the possibility that the individual may have had more than 1 genetically distinct strain; this was not demonstrated. The evidence presented from the RAPD analysis indicated that these individuals did not present as treatment failures but, instead, were compliance failures and had acquired a new infection between the treatment visit and the follow-up visit.
Trichomoniasis is the most common curable STD in the United States.2 In a recent survey of the US population, the overall prevalence in women was found to be 3.1%.11 The prevalence of trichomoniasis in inner-city US STD clinics typically approaches 25% and may be higher in certain populations.12,13 In Los Angeles, for instance, the prevalence among African American attendees at a public clinic was 38%.14 Trichomoniasis is also common in developing nations. Various studies of African populations have reported the prevalence of vaginal trichomoniasis to be between 11% and 25%.15–17 Laga et al.15 reported an incidence rate of 38% during a 4-month exposure interval among HIV-infected women in Zaire.
The current treatment of trichomoniasis is a single 2-g dose of either metronidazole or tinidazole; however, gastrointestinal intolerance is common for both metronidazole and tinidazole, preventing some women from adhering to the medication, especially if multiple doses are required.18,19 In addition, therapy with nitroimidazoles is associated with vaginal yeast infections as an adverse event.19 The actual rate of resistance of T. vaginalis to single-dose metronidazole is unclear but is likely around 5%.20,21 Strategies for treatment of such cases include increasing the doses of metronidazole or tinidazole as well as attempting cure with paromomycin intravaginal cream or boric acid vaginal suppositories, but none of these approaches are highly effective and, with the exception of boric acid, are poorly tolerated.7,22,23
The currently commercially available metronidazole gel with 37.5 mg of metronidazole per dose has been found to be only 50% efficacious for treating trichomoniasis.7 There is clearly a need for new therapeutic options for trichomoniasis, yet novel compounds do not seem to be on the horizon.
Although not composed of novel therapeutic agents, high-dose intravaginal metronidazole combined with miconazole offers the possibility of a well-tolerated treatment, which avoids the systemic adverse effects of nitroimidazoles. In this pilot study of effectiveness for trichomoniasis, we found the product to be as efficacious as oral metronidazole and well tolerated. Single-day dosing for 7 days was as effective as twice a day dosing in this small study. Although not studied, it is also possible that this regimen could be helpful in the treatment of metronidazole-resistant trichomoniasis due to the high local concentration of metronidazole. In addition, the presence of miconazole is a deterrent to the development of vaginal candidiasis as a result of treatment. Although not the primary objective of the study, it is also of interest that molecular techniques suggest that regimen, oral metronidazole, or metronidazole 750 mg/miconazole 200 mg was associated with treatment failure but rather that participants were reinfected during the course of the study. Limitations of the study include the small sample size, lack of nucleic acid amplification testing at the final study visit, and relatively short duration of follow-up. It is conceivably possible that the intravaginal medication simply suppressed, instead of entirely eliminating, the trichomonads. Although our study population was fairly homogenous in terms of race and age, there is no biologic reason to suspect that other populations would respond differently to the treatment under study.
In summary, intravaginal high-dose metronidazole 750mg/miconazole 200 mg for 7 days was as efficacious as single-dose metronidazole for the treatment of vaginal trichomoniasis in this small pilot study and may represent a viable alternative for treatment. Further studies looking at larger numbers of participants and length of therapy should be considered.
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