Bacterial vaginosis results from replacement of the hydrogen peroxide-producing Lactobacillus species which dominate the normal vaginal flora by various anaerobic and facultative bacteria.1,2 Its pathogenesis is not clearly understood. The diagnosis of bacterial vaginosis is not standardized. Various combinations of the four clinical Amsel criteria3 (clue cells on microscopic examination of vaginal discharge in saline; vaginal pH more than 4.5; thin, homogeneous, white-gray discharge; and a fishy odor after addition of potassium hydroxide) are commonly used in clinical practice. Card tests for alkalinity, trimethylamine, and proline aminopeptidase are also used. Gram stain4 provides a reproducible microbiologic diagnosis, but correlates with the widely used clinical criteria in approximately 80% of cases,5–7 and requires a delay in diagnosis. Variations in diagnostic criteria employed in clinical trials of treatments for bacterial vaginosis are widespread as well.
Approved therapies for bacterial vaginosis in the United States include oral and topical metronidazole, and topical clindamycin. Tinidazole (Tindamax, Mission Pharmacal Company, San Antonio TX, 1-[2-(ethyl sulfonyl) ethyl]-2-methyl-5-nitroimidazole) was recently approved in the United States for treatment of giardiasis, amebiasis, and trichomoniasis. Tinidazole is a second-generation nitroimidazole that has been used in Europe, Asia, and Latin America since the 1970s for giardiasis, trichomoniasis, amebiasis, bacterial vaginosis, gingivitis, Helicobacter pylori infections, surgical prophylaxis, and other anaerobic infections. Compared with metronidazole, a first-generation nitroimidazole, tinidazole has a twice-longer serum half-life (12–14 hours compared with 6–7 hours,) and side-effects have been reported to occur one half as often with tinidazole as with metronidazole.8,9 Easier reduction of the nitro group is thought to make tinidazole a more potent agent than metronidazole. Additionally, the increased lipid solubility of tinidazole results in more efficient penetration into both female and male reproductive tissues than metronidazole.10
Previous studies of tinidazole as a treatment for bacterial vaginosis have found favorable results. We undertook this randomized, multicenter, double-blind, double-dummy, placebo-controlled study to examine the effectiveness of tinidazole at 21–30 days of treatment, administered orally at 1 g once daily for 5 days and 2 g once daily for 2 days in 10 centers across the United States. Rigorous criteria for diagnosis and cure of bacterial vaginosis were applied as recommended in the 1998 U.S. Food and Drug Administration (FDA) Guidance Document entitled “Bacterial Vaginosis—Developing Antimicrobial Drugs for Treatment.”11
MATERIALS AND METHODS
Women seen in the clinics of the participating institutions, given a diagnosis of bacterial vaginosis, and referred for consideration of participation were candidates for this trial. All of the following criteria were required of candidates for enrollment: 1) age at least 18 years; 2) capable of providing written informed consent; 3) a negative pregnancy test on the day of enrollment; 4) able to follow the study protocol; 5) willing to forego coitus for days 1–10 of study participation, and to use nonlubricated condoms with each episode of coitus from day 11 until study completion; 6) willing not to douche or use any intravaginal products throughout participation, including tampons, medications, and devices; 7) willing to avoid drinking any alcohol from 24 hours before through 72 hours after taking study medication; and 8) premenopausal status. Further, the diagnosis of bacterial vaginosis, made by study personnel, required all five of the following: 1) 20% or more of vaginal squamous cells were clue cells on microscopic examination in saline; 2) a homogeneous, thin, white-gray vaginal discharge; 3) pH of vaginal discharge more than 4.5; 4) emanation of fishy odor from vaginal discharge after addition of 10% potassium hydroxide (positive whiff test); and 5) confirmation of vaginal bacterial morphotypes consistent with bacterial vaginosis on Gram stain (Nugent score 4 or more), as determined by a single reference laboratory.
The following characteristics excluded participants from participation: 1) symptoms so severe as to make assignment to placebo unacceptable to the patient; 2) identification of fungal elements or trichomonads by microscopic examination of vaginal discharge, lesions caused by herpes simplex virus or human papillomavirus on gross examination, or a positive nucleic acid amplification test for Chlamydia trachomatis or Neisseria gonorrhoeae; 3) presence of another vaginal, vulvar, or medical condition which might confound assessment of response to study treatment; 4) treatment with any 5–nitroimidazole, antifungal agent, or clindamycin within 14 days of study entry, or with any investigational drug within 30 days of study entry; 5) history of hypersensitivity to 5–nitroimidazole agents; 6) under treatment for cervical neoplasia during participation; 7) presently breastfeeding; 8) menstruating at diagnosis; 9) currently being treated with lithium or anti-coagulant drugs; 10) treatment with disulfiram within two weeks before, during, or within one week after study treatment.
The protocol and consent were approved by the institutional review board at each participating institution. Study personnel obtained written informed consent and Health Insurance Portability and Accountability Act of 1996 authorizations from candidates who qualified for participation using the above criteria. Demographic information, medical history, vaginal symptoms, vital signs, and physical examination data were collected. During pelvic examination, vaginal discharge samples were collected for establishing the diagnosis of bacterial vaginosis (including a sample that was heat-fixed on a microscope slide and submitted to a central laboratory for Gram stain interpretation), and culture for Candida spp and Trichomonas vaginalis. Endocervical swabbings were tested by the nucleic acid amplification platform in use at each site for C trachomatis and N gonorrhoeae. Samples for cervical cytology were obtained. If clinically indicated, samples for herpes simplex virus culture were obtained. Urine and blood samples were obtained for urinalysis, pregnancy test, complete blood cell counts and differential, and liver and kidney function measures.
Participants were instructed about the study protocol, given a daily diary in which to record drug administration, vaginal symptoms, any potential adverse events, and concomitant medications and given a supply of nonlubricated condoms. A telephone call was scheduled for 8–10 days after the first day of study drug administration.
All participants meeting all entry criteria and no exclusion criteria were randomly assigned to study treatment using the method of permuted blocks. Blocks were of size 3, one participant to each treatment, and randomization was conducted at each study center separately. Randomization was accomplished with SAS PROC PLAN (SAS Institute Inc., Cary, NC), which uses a prime modulus multiplicative congruential random number generator. Allocation of participants to study treatment was accomplished by assigning each consecutive participant to the next study number and corresponding drug packet, provided by the sponsor, in sequence at each center. All those randomly assigned make up the Intent-To-Treat population. To form the modified Intent-To-Treat population, those whose Gram stain score did not provide microbiologic confirmation of the diagnosis of bacterial vaginosis were omitted from the Intent-to-Treat population. The Per Protocol population excluded modified Intent-To-Treat participants who had other genital tract infections not apparent on entry visit examination, took less than 4 doses of study treatment, did not follow protocol, or withdrew or were withdrawn from participation before study completion.
The three treatment arms consisted of tinidazole 1 g (two 500-mg tablets) by mouth once daily for 5 days (Group I); tinidazole 2 g (four 500-mg tablets) by mouth once daily for 2 days (Group II); and placebo tablets by mouth once daily for 5 days (Group III). To preserve blinding, each participant received a packet containing 14 identically appearing tablets for 5 days: a day 1 bottle containing four tablets, a day 2 bottle containing four tablets, and day 3–5 bottles each containing two tablets.
At the day 8–10 telephone call, participants were queried about vaginal symptoms, compliance with protocol procedures, and any potential adverse events. The final visit was scheduled at 21–30 days after the first day of treatment. During the final visit medical history and vaginal symptoms data were collected, vital signs and physical and pelvic examinations were repeated, and vaginal discharge samples were taken to reexamine for diagnostic criteria and Gram stain interpretation. T vaginalis culture was also repeated. Urine and blood samples were taken to repeat the same testing done at the entry visit. If clinically indicated, retesting for chlamydia, gonococcus, and herpes virus was done. Diaries were reviewed with participants to ensure all adverse events were recorded on the case report forms.
Therapeutic cure was defined in accordance with the 1998 FDA Guidance Document. Therefore, a therapeutic cure was characterized by resolution of all five diagnostic criteria used to establish the diagnosis of bacterial vaginosis at study entry, which were 1) clue cells 20% or more of squamous cells; 2) homogenous, thin white-gray discharge; 3) vaginal pH more than 4.5; 4) positive whiff test; and 5) Nugent score 4 or more. Adverse events were classified for relationship to study treatment by investigators, and were considered serious if they caused death, were life threatening, required hospitalization, caused a persistent or significant disability, or caused congenital anomaly.
Because of potential variation among centers, the Cochran-Mantel-Haenszel procedure (which adjusts for such variation) was used to test for efficacy of the tinidazole treatments. A priori, the one-tailed 0.025 alpha level was chosen for testing. Because each tinidazole treatment was compared with placebo (two comparisons), the Bonferroni adjustment was applied, thus making the alpha level 0.0125, one-tailed. Sample size determination was accomplished using the normal approximation of the binomial distribution, assuming a 25% success rate for placebo and a 55% success rate for tinidazole; with one-tailed alpha 0.0125 and power 80%, at least 50 modified Intent-To-Treat participants per treatment arm were needed. The primary efficacy analysis of a therapeutic cure compared the two tinidazole regimens with placebo in the modified Intent-To-Treat population. Secondary efficacy analyses using the same statistical method examined cure rates defined by resolution of four and three or more clinical (Amsel) criteria and microbiologic (Gram stain) criteria individually.
Ten geographically diverse centers in the United States participated, screened 379 candidates, and qualified and enrolled 235 participants from February 1, 2005, to January 11, 2006. Table 1 shows the disposition of participants by treatment arm. Centers enrolled 11–43 participants each. Table 2 shows that participants were evenly distributed among the three treatment arms, and that age, height, weight, ethnicity, entry Gram stain score, and vaginal pH were comparable as well. The mean Gram stain scores at entry in all three treatment arms were high (8.39, 8.24, and 8.35 in Groups I, II and III, respectively), as expected among women with clinical signs. Participants classified with recalcitrant bacterial vaginosis (three or more prior episodes within the previous 12 months) were comparable among the groups (19.2%, 20.0%, and 24.7% in Groups I, II, and III, respectively). Compliance with study therapy based on tablet count was 92.9%, 88.5%, and 89.1% for the three respective groups. Table 3 shows superior efficacy of both tinidazole regimens to placebo in the modified Intent-To-Treat population (primary endpoint), as well as in the Intent-To-Treat and Per Protocol populations when cure is defined by normalization of all four Amsel criteria and Gram stain score, by Gram stain score less than 4, and by resolution of four and three or more Amsel criteria. When normalization of the individual Amsel criteria by treatment arm for each population was examined, results very similar to those for resolution of three of four Amsel criteria were found (data not shown).
Adverse events (Table 4) occurred with comparable frequency in tinidazole and placebo recipients, except for dysgeusia, which was significantly more common in the tinidazole arms, and nausea, which was significantly more frequent in Group II. However, no difference was seen between the tinidazole and placebo groups in the number of participants experiencing one or more gastrointestinal symptoms. Comparing the proportion of participants experiencing any adverse event(s) in Group I (55.8%) and Group II (61.5%) to the placebo group (48.8%) by Fisher exact test gave values of P=.426 and P=.113, respectively. One serious adverse event, a perineal abscess in a placebo recipient, required withdrawal of the patient from the study. No patient withdrew because of unacceptability of tinidazole treatment. No clinically significant effect of tinidazole was seen on blood cell counts, liver or kidney function tests, or urinalysis results.
We are aware of 21 published clinical trials9,12–31 that have evaluated the efficacy of tinidazole in the treatment of bacterial vaginosis; most have been done outside the United States. Trial regimens range from 1–2 g daily for 1–5 days. All have found favorable results. Our present study, performed at 10 centers across the United States, demonstrates the efficacy and tolerability of tinidazole, both 1 g once daily for 5 days and 2 g once daily for 2 days, in the treatment of bacterial vaginosis when compared with placebo.
In trials evaluating treatments for bacterial vaginosis it has been common for studies to use some or all of the Amsel criteria, Gram stain results, or weighted combinations of the two to define diagnosis and cure. In 1998, the FDA issued a Guidance Document entitled “Bacterial Vaginosis—Developing Antimicrobial Drugs for Treatment,”11 which specifically called for investigators to define bacterial vaginosis in study participants as presence of all four Amsel criteria and a Gram stain score of 4 or more, to define cure as the absence of all four Amsel criteria and a Gram Stain score of 0–3, and to perform the examination for evaluation of therapeutic response at 21–30 days after the first day of treatment. Thomas et al32 are among few who have previously acknowledged these guidelines. They conclude that the rigor of the criteria can cause significant differences in treatment outcomes to be missed. Specifically, elimination of anaerobic overgrowth with consequent clearance of odor (amines) and clue cells is not appreciated by these criteria in participants who do not also reestablish Lactobacillus predominance to achieve a pH decrease. They note also that pH decrease does not correlate with greater durability of cure in existing literature. Our study confirms an improved cure rate when 3 of 4 Amsel criteria are used to define cure, but we did not find that eliminating the requirement for pH less than 4.5 yielded a higher cure rate than eliminating requirements for clue cells less than 20% or negative whiff test (data not shown).
We elected to use consistently the most rigorous criteria for the primary efficacy endpoint in our study. Specifically, we followed precisely the recommendations of the 1998 FDA Guidance Document, and applied them to the entire study population (modified Intent To Treat) meeting the diagnostic criteria. Using those methods we found that therapeutic cure rates of bacterial vaginosis for the 1 g once daily for 5 days group (36.8%, number needed to treat 3.2) and for the 2 g once daily for 2 days group (27.4%, number needed to treat 4.5) both reached high statistical significance in comparison with placebo (5.1%). Using the more traditional definitions of cure based only on clinical criteria predictably produced substantially higher cure rates for tinidazole (Table 3).
In the interest of our participants, we designed our protocol to exclude those with symptoms too severe to tolerate assignment to placebo, raising the possibility that only those with milder symptoms might be included in our study. Because our participants met all four Amsel criteria and Gram stain criteria for diagnosis, their Gram stain scores were high, and no patient at any center was excluded because of severe symptoms, we do not believe our findings are limited to milder cases.
Comparison of (particularly quantitative) results from one trial to those of another often leads to erroneous conclusions because of differences in study design, enrollment and outcome criteria, data collection, test methodology, selection of participants to include in the analyses, and a plethora of other seen and unseen confounders. Treatment trials for bacterial vaginosis are especially subject to such errors because of variations in the definitions of diagnosis and cure. However, because application of the FDA Guidance Document criteria effectively resets the reference standard for efficacy of therapy of bacterial vaginosis, we feel it may be useful to juxtapose our results to those available from trials of other agents which have used those criteria. These data are shown in Table 5.
Tinidazole therapy was well tolerated in this trial, as evidenced by the high compliance with therapy in both treatment arms. Compared with standard metronidazole given as 500 mg twice daily for 7 days, tinidazole therapy for bacterial vaginosis can reduce the total number of doses necessary by more than half. Additionally, the low incidence of posttreatment vaginal candidiasis seen in both tinidazole treatment arms compares favorably with current FDA-approved intravaginal and oral therapies. Finally, any gastrointestinal symptom(s) were seen with equal frequency in the tinidazole and placebo groups, and the incidence of nausea (a frequent complaint with oral metronidazole therapy) in the 1 g daily tinidazole treatment arm was not significantly different from that observed in the placebo arm.
Tinidazole by mouth, 1 g once daily for 5 days or 2 g once daily for 2 days, is an effective treatment for bacterial vaginosis in our rigorous therapeutic trial, and is competitive with available treatments. The 1 g once daily for 5 days regimen showed trends toward better efficacy and tolerability than the 2 g once daily for 2 days regimen. Although our study was not adequately powered to prove safety, we discovered no safety concerns with this use of tinidazole.
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