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Why Do Different Criteria for ‘Cure’ Yield Different Conclusions in Comparing Two Treatments for Bacterial Vaginosis?

Thomas, Katherine K. MS*; Sanchez, Sixto MD, MPH†; Garcia, Patricia J. MD, MPH‡; Holmes, King K. MD, PhD*

Commentary

Objective: The objective of this study was to determine why different criteria for response to treatment of bacterial vaginosis (BV) led to markedly different conclusions about treatment efficacy in a randomized trial comparing metronidazole gel versus metronidazole/nystatin ovules.

Study: We compared the impact of two treatment regimens on individual components of Amsel and Nugent criteria at follow-up visits 14, 42, and 104 days after initiating treatment.

Results: Compared with gel, ovules more effectively eliminated amines, clue cells, and Gardnerella, Prevotella, or Mobiluncus morphotypes from vaginal fluid, thus achieving cure based on “usual” criteria (absence of BV by Amsel or Nugent criteria), but did not more effectively restore Lactobacillus morphotypes or lower vaginal pH, thus not meeting Federal Drug Administration (FDA) criteria for cure.

Conclusion: Because early vaginal recolonization by lactobacilli was poor after both gel and ovules, FDA draft criteria for cure missed marked differences in treatment efficacies against Gardnerella, clue cells, and amines. Cure defined more “usually” may give more useful information.

Federal Drug Administration guidance criteria for cure of bacterial vaginosis include rapid vaginal recolonization by lactobacilli, which many women lacked even when bacterial vaginosis-associated bacterial morphotypes were eliminated after intravaginal metronidazole treatment.

From the *University of Washington Departments of Medicine and the Center for AIDS and STD, Seattle, Washington; †Hospital dos de Mayo, Lima, Peru; and the ‡School of Public Health and Administration, Universidad Peruana Cayetano Heredia, Lima, Peru

*Originally made by Litmus Concepts and now sold in the United States as the FemExam Card Test by Cooper Pharmaceuticals and as the Quickview ADVANCE pH and amines test by Quidel Corp.

The authors thank Jeanne Marrazzo, MD, for advice on preparation of the manuscript.

Supported by the University of Washington STD Cooperative Research Center (AI-31448) and 3M Pharmaceuticals.

Correspondence: King K. Holmes, MD, PhD, Director, UW Center for AIDS and STD, University of Washington, MS #359931, 325 9th Ave, Seattle, WA 98104. E-mail: worthy@u.washington.edu.

Received for publication October 15, 2004, and accepted February 4, 2005.

AFTER THE INTRODUCTION OF Amsel criteria1 for the evaluation of bacterial vaginosis (BV), many treatment trials reported outcomes in terms of posttreatment detection of BV by Amsel criteria2–20 (Table 1). The Amsel criteria for BV require the presence of any three of the following four criteria: 1) homogeneous vaginal discharge, 2) vaginal fluid pH >4.5, 3) release of fishy amine odor from vaginal fluid when mixed with 10% KOH solution, and 4) presence of clue cells in vaginal fluid (representing at least 20% of cells in a commonly used modification21). A commonly reported successful posttreatment outcome has been absence of BV by Amsel criteria (i.e., <3 criteria present). However, individual studies have varied greatly in what formulation of the Amsel criteria and other measures are considered, including the Nugent criteria, consisting of a Gram stain score of 7 or more. The Gram stain score varies from 0 to 10 and is computed from concentrations observed on vaginal fluid Gram stain of Gardnerella, Prevotella and Mobiluncus, and Lactobacillus morphotypes.22

The current U. S. Food and Drug Administration (FDA) Draft Guidance for BV cure in a treatment trial, published for comment in 1998 but still considered a Draft Guidance, calls for one test of cure (TOC) visit at 21 to 30 days after starting treatment, and defines therapeutic cure as clinical cure (0 of the 4 Amsel criteria) and Nugent score of 0 to 3. Since then, some reports have provided outcomes based both on Nugent score <4 (normal) and <7 (not consistent with BV) along with outcomes based on Amsel criteria.8,12,13,23,24 However, few if any published evaluations of treatment for BV have defined outcomes based on cure defined by the FDA Draft Guidance criteria. Most recent publications8,12,13,16,23–25 continue to report treatment outcomes using less rigorous criteria involving Amsel criteria but not Nugent score, as in pre-1998 studies.

In our recent comparison of two intravaginal metronidazole-containing regimens for the treatment of BV, we observed a significant difference between the two regimens when we defined BV by the presence at follow up of at least three of four Amsel criteria, of a Nugent score consistent with BV, or both, but found no significant difference in rates of cure as defined by the 1998 FDA Guidance. As separately reported,26 at follow up 10 to 21 days after starting treatment, BV was detected by Amsel and Nugent criteria in 2% of subjects (one of 51) given ovules containing metronidazole and nystatin versus 23% (13 of 57) given gel containing metronidazole (P <0.01). However, rates of cure per FDA Draft Guidance criteria were 35% (18 of 51) in those given ovules versus 26% (15 of 57) in those given gel (P = 0.4). To determine the reason(s) for these disparate results, we analyzed the effects of these two treatment regimens on the various components of the Amsel and Nugent criteria.

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Materials and Methods

Data were collected from a trial of two intravaginal metronidazole treatments carried out at Dos de Mayo Hospital in Lima, Peru, from February 19, 1999, to April 24, 2000. Recruitment and randomization are described elsewhere.26 Briefly, women 18 to 40 years old and reporting vaginal discharge or vaginal malodor were recruited for the study. Women were tested for pH ≥4.7 and amines in vaginal fluid using a point-of-care test,* appearance of vaginal discharge was noted, and vaginal fluid was collected for Gram stain and microscopy after mixing separately with saline and with 10% potassium hydroxide. Women with vaginal fluid positive for both pH ≥4.7 and for amines were randomly assigned to one of two intravaginal treatments: metronidazole 0.75% gel (an FDA- approved treatment for BV, delivering 37.5 mg metronidazole per nightly dose) or ovules containing 500 mg metronidazole plus 1,000 U nystatin (Flagystatin, intended for syndromic management of vaginal discharge and a commonly prescribed medication for BV symptoms in Peru26). Both regimens were prescribed nightly for 5 nights.

Women were asked to return at 14 days, 42 days, and 104 days after the baseline visit for speculum examination, pelvic examination, and examination of vaginal fluid for pH ≥4.7, amines, appearance, and saline/KOH microscopy and Gram stain. Women meeting Amsel criteria were given treatment for BV at that follow-up visit. Women whose Nugent score was subsequently read as consistent with BV7–10 were given treatment for BV at their next scheduled follow-up visit. The treatment code was broken only after all cases had completed follow up, outcomes at all visits had been classified according to Nugent score and Amsel criteria, and data analyses comparing the two arms for presence of BV (persistence or recurrence) were completed.

For purposes of analyses, the diagnosis of BV at baseline was based on a Nugent score of 7 to 10 plus at least three Amsel criteria: vaginal fluid pH ≥4.7 and positive amine test, plus either homogeneous vaginal discharge or clue cells representing more than 20% of cells in vaginal fluid.

Two definitions of “cure” for the two metronidazole-containing treatments were considered at 10 to 21 days after initiation of treatment: cure by “usual” criteria, defined to be absence of BV by widely reported criteria (<3 Amsel criteria or Nugent score <7); and cure by FDA guidance criteria, defined on the basis of finding none of the four Amsel criteria (clinical cure) and a Nugent score of <4. Because we asked women to return at 14 days after initiating treatment, we could not examine the outcomes at 21 to 30 days, as recommended by the FDA Draft Guidance, but we also analyzed data of all women returning for first follow up at any time after treatment. We then examined the effect of each treatment on each of the individual components of the Amsel criteria and on the components of Nugent’s score.

Comparisons between arms at the first follow-up visit were performed by Fisher’s exact tests.

We also examined whether any outcomes at the first follow up predicted subsequent recurrence of BV by both Amsel and Nugent criteria by analyzing data from second and third follow-up visits among women who lacked BV by both criteria at the first follow-up visit. Variables collected at first follow up, which were considered for prediction of subsequent recurrence of BV, included: 1) overall cure by FDA guidance and 2) individual components of these criteria for cure, including pH <4.7, negative amine test, absence of Gardnerella, Prevotella, or Mobiluncus morphotypes on Gram stain of vaginal fluid, presence of Lactobacillus morphotypes, and Nugent score <4. Fisher exact tests were used to assess whether these components of cure at the first follow-up visit were predictive of subsequent development of BV by both Amsel and Nugent criteria at the second follow-up visit. Survival analysis, which included both second and third follow-up visits, was used to confirm that results were similar when all visits were analyzed.

Analyses were performed in S-Plus 2000 professional release 2, 1999.

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Results

Of 184 women randomized, 151 had BV by both Amsel and Nugent criteria at baseline, of whom 138 (91%) returned for at least one follow-up visit (108 within 10–21 days), 120 (79%) for a second, and 75 (50%) for a third follow-up visit. The median (range) interval from baseline visit was 15 days (range, 10–132 days) to the first follow-up visit, 56 days (range, 38–218 days) to the second follow-up, and 127 days (range, 82–298 days) to the third follow-up visit.

Rates of cure per the FDA Guidance criteria were 35% (18 of 51) in those given metronidazole/nystatin ovules versus 26% (15 of 57) in those given metronidazole gel (P = 0.4) (Table 2).

Examining the specific components that make up the FDA Guidance criteria for cure revealed that metronidazole/nystatin ovules (delivering 500 mg metronidazole per night) were more effective than metronidazole gel (delivering 37.5 mg metronidazole per night) in reducing Gardnerella or Prevotella morphotypes to undetectable by Gram stain (P = 0.01), in eliminating clue cells to less than 20% of all vaginal epithelial cells (P <0.01), and in eliminating amines (P <0.01) at 10 to 21 days. Mobiluncus morphotypes were uncommon, but considering all first follow-up visits rather than only those ≤21 days, ovules were also more effective at reducing Mobiluncus morphotypes to undetectable (P = 0.06, data not shown).

However, ovules and gel treatments did not differ significantly in terms of effecting the reappearance of Lactobacillus morphotypes in Gram stain of vaginal fluid: at 10 to 21 days, Lactobacillus morphotypes were observed in 65% in the ovule arm versus 51% in the gel arm (P = 0.2), and 45% versus 30% had Lactobacillus morphotypes >30/100× microscopic field in these arms, respectively (P = 0.1). The two treatments also resulted in no significant difference in vaginal pH <4.7 seen in 59% of patients given ovules and 42% of patients given gel (P = 0.1). Absence of Lactobacillus morphotypes in Gram stain of vaginal fluid at 10 to 21 days was significantly correlated with pH ≥4.7 (P <0.001, Table 3). There was no difference between the two arms at 10 to 21 days in prevalence of homogeneous discharge consistent with BV, but this was uncommon in both arms.

In the absence of the criteria for BV at the first follow-up visit, we saw no evidence that failure of Lactobacillus morphotypes to appear on vaginal Gram stain predicted subsequent development of BV. Combining both arms of the study, among those without BV as defined by both Amsel and Nugent criteria at the first follow-up visit (or treated again with metronidazole at that visit), BV by both Amsel and Nugent criteria was detected at the second follow-up visit in 13 (22%) of 60 with Lactobacillus morphotypes versus six (22%)-of 30 without Lactobacillus morphotypes seen at the first follow-up (relative risk [RR] = 0.9, 95% confidence interval [CI] = 0.4–2.2); in 11 (22%) of 49 with pH <4.7 versus eight (20%) of 41 with pH ≥4.7 at the first follow up (RR = 0.9, 95% CI = 0.4–2.0); in 14 (19%) of 75 with no Gardnerella morphotypes versus 5 (33%) of 15 with such morphotypes at the first follow-up (RR = 0.6, 95% CI = 0.2–1.3); and in four (13%) of 30 who met FDA Guidance criteria for BV cure versus 15 (25%) of 60 who did not meet these criteria at the first follow up (RR = 0.5, 95% CI = 0.2–1.5). Similarly, no significant differences were noted for subanalyses within either arm, although the numbers of subjects within each arm were small. Survival analyses of both second and third follow-up visits yielded similar results to those of the second follow-up visits.

Follow-up at the interval recommended in FDA Guidance criteria did not increase the prevalence of Lactobacillus morphotypes on vaginal fluid Gram stain. Examining only first follow-up data, prevalence was 57% in 108 who returned for first follow up in 10 to 21 days (our follow-up interval) and 61% in 18 returning in 21 to 30 days (FDA Guidance follow-up interval). In addition, we saw no evidence that longer follow-up was associated with a higher likelihood of detecting Lactobacillus morphotypes. Of 64 women who did not meet both Amsel and Nugent criteria for BV or receive treatment for BV at 10 to 21 days, and who also returned for follow-up at 42 to 90 days, prevalence of Lactobacillus morphotypes was actually lower at the later visit, having fallen from 66% at 10 to 21 days to only 48% at 42 to 90 days.

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Discussion

In this study, intravaginal ovules containing 500 mg metronidazole and 100,000 U nystatin ovules were more effective than gel delivering 37.5 mg metronidazole in eliminating abnormal bacterial morphotypes and the related clue cells and amines associated with BV, but were not significantly more effective as measured by enhanced detection of vaginal lactobacilli as assessed by Gram stain. Absence of Lactobacillus morphotypes on Gram stain and pH ≥4.7 were highly correlated with one another and were commonly seen in both arms after treatment. The absence of a substantial difference between the two treatments in effecting an increase in vaginal lactobacilli as detected by Gram stain and in frequency of abnormal vaginal pH at 10 to 21 days explained why the ovules were not more effective than the gel in reducing the number of Amsel criteria present to 0 or the Nugent score to <4, even though they were significantly more effective in reducing the Amsel criteria to <3 and the Nugent score to <7.

The 1998 FDA Draft Guidelines for demonstrating cure for BV have not been widely adopted since they were published. We hypothesize that this may be because many researchers have been unaware of them and because cure rates using the guidelines are so poor. Whether the FDA Draft Guidelines should be widely adopted is important to consider. In our study, the FDA criteria for cure give dramatically lower rates than the “usual” criteria for cure. Agreement on what to use must be reached so that studies may be compared with one another. However, which is better? Our study demonstrates that the FDA criteria for cure could completely miss differences between regimens with markedly different efficacies against Gardnerella, clue cells, and amines when there is no substantial difference in promoting rapid vaginal recolonization with lactobacilli. In this example, we would have seen no difference between the two treatments if we had relied solely on cure defined by FDA Draft Guidance criteria. Our observation that combining both treatment arms, absence of Lactobacillus morphotypes at the first posttreatment follow up did not predict recurrence of BV between the first and second follow-up visits suggests that the early return of lactobacilli and resulting in lowering of vaginal pH may not be an essential component of a durable cure. If this proves to be the case, then cure as more usually defined based on Amsel criteria or Nugent score may give more useful information than does cure as defined by FDA Guidance. However, longer posttreatment follow-up of larger numbers of patients would be required to examine this question.

Other studies have reported higher prevalences of Lactobacillus morphotypes on Gram stain after treatment with standard doses of intravaginal or oral metronidazole.9,27 Most of our patients returned between 10 and 21 days, rather than in the 21- to 30-day window recommended in the 1998 FDA Draft Guidance for the test of cure visit. However, we saw no evidence that a later first follow-up visit would have increased the prevalence of Lactobacillus morphotypes seen. A lack of significant increase in prevalence of Lactobacillus morphotypes on Gram stain with posttreatment follow up longer than 21 days has also been seen in other studies. Agnew et al. found 94% and 91% with Lactobacillus morphotypes on Gram stain at >35 days after treatment with intravaginal metronidazole gel and oral metronidazole, respectively, but in the same study, prevalence after each treatment was already high (83%) at ≤21 days.27 In asymptomatic women diagnosed with BV by Nugent criteria, Schwebke found that 14 days after initiating treatment with metronidazole gel, 38% of 26 women had pH ≤4.5, and only 36% of 25 of the same women had pH ≤4.5, 2 or more weeks later.23 In fact, we have not found published evidence for a significant increase in Lactobacillus morphotypes among women followed for longer than 21 days. The prevalences of Lactobacillus morphotypes in baseline vaginal Gram-stained smears in BV treatment studies have varied in the literature from 5%28 to 68%.29 In our study of 151 subjects with BV by both Nugent and Amsel criteria, only 3% had Lactobacillus morphotypes detected in vaginal fluid at baseline.

Limitations of this study include the variability of elapsed time between visits, related in part to aggressive efforts to bring back participants who had missed earlier appointments. Although 91% of women returned for at least one follow-up visit, only 72% did so within 21 days after initiating treatment. Results of comparisons between arms were very similar whether all first follow-up visits were considered or only those within the first 21 days. Other limitations include the lack of Lactobacillus culture and inability to define whether hydrogen peroxide-producing lactobacilli were present. Although neither the FDA nor usual criteria for cure include culture for Lactobacillus or define whether lactobacilli seen are hydrogen peroxide-producing, vaginal cultures might have detected lower concentrations of lactobacilli not easily detected by Gram stain, and presence of H2O2-producing lactobacilli might predict subsequent recurrence even if presence of lactobacilli detected on Gram stain do not.

In conclusion, although the higher dosing of metronidazole delivered by the metronidazole plus nystatin ovules for topical therapy of BV as a 5-day course appears to have had a significantly greater impact than metronidazole gel in reducing the posttreatment persistence of Gardnerella morphotypes, amines, and clue cells, this regimen may not be sufficient to effect rapid vaginal recolonization by Lactobacillus in many women treated for BV. Future treatment trials should disaggregate outcomes that include impact on signs, abnormal bacterial flora, and recolonization by H2O2-producing lactobacilli. To choose between “usual” and FDA Guidance criteria for cure, researchers must consider whether criteria for cure of BV should include early restoration of Lactobacillus-dominant vaginal flora with resulting lowering of vaginal pH, like the FDA Guidance criteria do. Whether the FDA criteria should be widely adopted may depend on whether the longer-term prognosis for return of symptoms or complications of BV ultimately proves to be related to these indicators.

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