JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 2010, p. 1317–1322 0095-1137/10/$12.00 doi:10.1128/JCM.00359-09 Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Vol. 48, No. 4

Randomized, Double-Blind Crossover Study of Vaginal Microflora and Epithelium in Women Using a Tampon with a “Winged” Apertured Film Cover and a Commercial Tampon with a Nonwoven Fleece Cover䌤 David J. Chase,1* Berenike P. Schenkel,2 Anne-Marie Fahr,3 and Ulrich Eigner4 for the Tampon Study Group† Scientific and Medical Affairs, Johnson & Johnson Consumer and Personal Products Worldwide, 199 Grandview Road, Skillman, New Jersey 085581; European Medical Affairs, Johnson & Johnson GmbH, Kaiserswerther Str. 270, 40474 Du ¨sseldorf, Germany2; Department of Microbiology and Hygiene, Laboratory Limbach, Im Breitspiel 15, 69126 Heidelberg, Germany3; and Department of Microbiology and Hygiene, Laboratory Limbach, Im Breitspiel 15, 69126 Heidelberg, Germany4 Received 19 March 2009/Returned for modification 21 December 2009/Accepted 5 February 2010

This study compared the safety of a new tampon with a four-winged apertured film cover over its nonwoven cover to improve leakage performance with that of a commercial tampon with a nonwoven cover only. Healthy women (evaluable, n ⴝ 69) were randomized to crossover between test and reference tampons in two consecutive menstrual cycles. Qualitative and quantitative analyses of vaginal cultures were conducted pre-, mid-, and postmenstrually for a broad panel of microorganisms, and colposcopy was performed. Similar to previous studies, prevalence and mean colony counts of the majority of microorganisms generally increased midmenstrually and returned or began to return postmenstrually. In contrast to most previous studies, Lactobacillus species remained at similar levels throughout the cycles with both tampons. Neither tampon was associated with clinically significant microbiological changes or abnormalities or with vaginal/cervical epithelial integrity changes on colposcopy. Microbiological and colposcopic evaluations indicate that the apertured film-covered tampon is safe.

keeping with the European General Product Safety Directive (4) and the European Code of Practice for Tampons (defined by the European Disposables and Nonwovens Association, an international trade association of absorbent hygiene product industries that, among other things, establishes standards for hygiene products) (5). Until about 5 years ago, all marketed tampons that had covers over their absorbent cores (which consist of rayon, cotton, or blends of these fibers) had lightweight, nonwoven fleece as the cover material. In 2003, the first tampon with an apertured film cover was introduced (o.b. Comfort from Johnson & Johnson; initially introduced in Germany and subsequently in other European countries as well as in the United States) after being shown in consumer studies to provide noticeable benefits with respect to ease and comfort of use (insertion and removal, in particular). A clinical study was conducted to demonstrate that this new film-covered tampon was as safe as its fleececovered predecessor and did not differ with respect to effects on either the vaginal epithelium or the vaginal microflora (1). Further studies of the film-covered tampon—in vitro and in vivo—revealed that if the apertured film was applied to the absorbent core of the tampon in such a way that areas of the film not directly attached to the core were gathered into double-layered folds (“wings”), the tampon absorbed fluid more effectively than the predecessor film- or fleece-covered tampons (Johnson & Johnson Consumer & Personal Products Worldwide, unpublished data). It appeared that two or more layers of wings of apertured film, closely apposed to each other, could act as “parallel plates,” conducting fluid to the absorbent core of the tampon. Thus, when four such wings were formed, the tampon came to have multiple sets of parallel

Commercial intravaginal menstrual tampons are used commonly by approximately one-half to two-thirds of women in the industrialized world. Since their introduction in the 1930s, the acceptance of tampons has been supported by demonstrations of their safety in clinical studies involving microbiological analyses, gynecological examinations, and subjects’ evaluations (1, 6, 13, 14). Vaginal microflora studies conducted at various time points during the menstrual cycle have shown tampons to have no significant effect on the qualitative or quantitative changes in either the aerobic or anaerobic organisms that occur normally during menses (2, 7–11, 15, 16, 18). Regulatory requirements for tampons differ among countries, with the United States, Canada, and Australia having some of the most rigorous regulations. The U.S. Food and Drug Administration (FDA) (17), for example, classifies tampons as medical devices (class II, special controls) and requires preclinical and clinical function and safety studies both for new tampon products and for significant changes in the design or the material of commercially available products. In contrast, countries such as those in the European Union classify tampons as commodities or consumer products and do not require clinical studies for marketing. In Europe, each manufacturer determines whether a clinical trial is necessary to confirm the safety of significant changes in tampon materials or design in * Corresponding author. Mailing address: Johnson & Johnson Consumer and Personal Products Worldwide, 199 Grandview Road, Skillman, NJ 08558. Phone: (908) 874-2524. Fax: (908) 874-1193. E-mail: [email protected]. † Members of the Tampon Study Group are listed in the Acknowledgments section of this article. 䌤 Published ahead of print on 17 February 2010. 1317

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plates conducting fluid to different areas (“absorbent fronts”) of the tampon’s core, thereby improving the ability of the tampon structure to manage and absorb fluid without increasing its overall capacity or absorbency. Consumer studies showed that this tampon reduced the incidence of “bypass” leakage of menstrual fluid (leakage before the tampon has become saturated) compared with other tampons, especially among women with heavier menstrual flow who were prone to experience such leakage. The present study was undertaken to compare the safety of a newly designed tampon that has a four-winged apertured film cover over its nonwoven cover with a commercially available tampon having a nonwoven cover only, with particular consideration of the vaginal microflora and the integrity of vaginal and cervical surfaces. MATERIALS AND METHODS The study design, including subject inclusion/exclusion criteria and study procedures/assessments, duplicates that of a similar study published previously (1). In brief, this was a double-blind, randomized, two-way crossover study comparing the current commercially available reference tampon (o.b. Normal with a nonwoven cover) with the test tampon (o.b. Normal with a four-winged apertured film cover heat-sealed to a nonwoven cover). The study was conducted at three centers in Germany according to FDA regulations and the International Conference on Harmonisation Guidelines for Good Clinical Practice. All subjects were fully informed and provided written consent for participation prior to enrollment. Study participants were healthy women 18 to 45 years of age with regular menstrual cycles of 21 to 35 days who were regular users of o.b. Normal absorbency tampons or other brands of similar absorbency for at least 4 days of their menses. Eligible subjects were randomly assigned (computer-generated randomization schedule) to use the two tampon products in one of two sequences: (i) cycle 1, test tampon, and cycle 2, reference tampon (test-reference group); (ii) cycle 1, reference tampon, and cycle 2, test tampon (reference-test group). The study consisted of an initial screening visit followed by assessment visits through two consecutive menstrual cycles. Three visits were performed during each menstrual cycle: a premenstrual visit during days ⫺10 to ⫺1 (with day 1 representing the first day of menstruation); a midmenstrual visit during days 2 to 4, after two or more tampons had been used for a total of at least 12 h; and a postmenstrual visit during days 7 to 12, at least 48 h after the last tampon was used. If complete data were not available for any one cycle and/or the subject was not able to complete a cycle, the investigator could allow the cycle to be repeated at the end of the study (cycle 3) using the appropriate tampon. A subject was withdrawn from the study if any data were missing for two cycles or if she did not comply with study tampon use for two cycles. At screening, vaginal swabs were obtained for Gram staining and quantitative culture to determine the presence of bacterial vaginosis. At the pre-, mid-, and postmenstrual visits, the following microorganisms were identified and evaluated quantitatively by methods described previously (1): Lactobacillus species, Staphylococcus aureus, Escherichia coli, group B Streptococcus, Gardnerella vaginalis, anaerobic Gram-negative rods, Enterococcus species, Candida albicans, and Candida species. Vaginal pH was measured at pre- and postmenstrual visits using pH strips (Macherey-Nagel, pH Fix 3.6-6.1). Because these measurements were based on orange-red color changes of pH paper, they could not be done reliably during menses. Details of statistical analyses and sample size calculations are also available in the previous publication by Chase et al. (1). In brief, no single microorganism or other variable formed using a combination of single microorganisms was considered to be the primary clinical outcome. Although the analysis and interpretation of results were mostly descriptive, a variety of statistical tests were performed. Most analyses of data from this balanced, crossover trial were conducted on the per-protocol population (defined as subjects who completed the study); adverse events were analyzed using the intent-to-treat (ITT) population (defined as subjects who used at least one of the study tampons).

RESULTS Demographic characteristics and subject disposition. In all, 80 women were enrolled in the study for two menstrual peri-

TABLE 1. Demographic characteristics of the intent-to-treat population Result for treatment sequence: Testreference (n ⫽ 41)

Referencetest (n ⫽ 39)

Total (n ⫽ 80)

29.9 ⫾ 7.87 (19–45)

29.3 ⫾ 8.20 (18–45)

29.6 ⫾ 7.99 (18–45)

No. (%) of women with menstrual flow: Heavy Moderate Light

3 (7.3) 38 (92.7) 0 (0.0)

0 (0.0) 39 (100.0) 0 (0.0)

3 (3.8) 77 (96.3) 0 (0.0)

No. (%) of women with absorbency of tampon normally used ofa: 1 droplet 2 droplets 3 droplets 4 droplets 5 droplets 6 droplets

5 (12.2) 13 (31.7) 41 (100.0) 10 (24.4) 2 (4.9) 0 (0.0)

5 (12.8) 13 (33.3) 39 (100.0) 6 (15.4) 0 (0.0) 0 (0.0)

10 (12.5) 26 (32.5) 80 (100.0) 16 (20.0) 2 (2.5) 0 (0.0)

Characteristic

Mean ⫾ SD age in yr (range)

Mean ⫾ SD no. of days/cycle (range)

28.1 ⫾ 0.85 (24–30)

27.8 ⫾ 1.0 (24–30)

27.9 ⫾ 0.93 (24–30)

Mean ⫾ SD no. of days of flow (range)

4.8 ⫾ 0.79 (4–8)

4.8 ⫾ 0.72 (4–6)

4.8 ⫾ 0.74 (4–8)

a More than one reply was possible. Findings are based on the absorbency classification of the European Disposables and Nonwovens Association Code of Practice for Tampons (5): 1 droplet, ⬍6 g; 2 droplets, 6 to 9 g; 3 droplets, 9 to 12 g; 4 droplets, 12 to 15 g; 5 droplets, 15 to 18 g; and 6 droplets, 18 to 21 g. Droplet categories 1 to 5 correspond roughly with FDA labeling designations for tampon absorbencies in the United States (16): 1 droplet, “Junior”; 2 droplets, “Regular”; 3 droplets, “Super”; 4 droplets, “Super Plus”; and 5 droplets, “Ultra.” Absorbencies higher than 18 g are not covered by U.S. regulations.

ods. Enrollment started in March 2005, and the last subject completed the study in June 2005. A total of 41 women were randomized to the test-reference group and 39 to the reference-test Group. All enrollees in both groups were Caucasian, and age distributions were similar across treatment sequences (Table 1). There was little difference between groups (across treatment sequences) in menstrual flow, cycle length, absorbency of tampons usually used, and duration of flow (Table 1). Among all subjects (ITT population), 58.8% (n ⫽ 47) used oral contraceptives and 8.8% (n ⫽ 7) used intrauterine devices, while 15% (n ⫽ 12) used no contraception at all. All 80 enrolled subjects completed the study. Major protocol violations were diary entries that did not match the case report form (n ⫽ 4); tampon noncompliance (n ⫽ 3); diary entries that were questionable (n ⫽ 2); and interruption in tampon use of more than 30 min, antibiotic comedication, postmenstrual visit out of the time window, or colpitis within 2 months prior to the study (n ⫽ 1 for each). The per-protocol population (n ⫽ 69 subjects) included 36 subjects in the test-reference group and 33 in the reference-test group. Microbiology results. Table 2 summarizes microbiology results and provides comparisons within and between tampons. Sequential changes in vaginal microflora during menstrual cycles. Midmenstrual prevalence of potentially pathogenic mi-

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Microorganism

Lactobacillus species S. aureus E. coli Group B Streptococcus G. vaginalis Anaerobic Gram-negative rods Enterococcus species C. albicans Candida species Colony count (mean ⫾ SD)c

68 (98.6) 26 (37.7) 31 (44.9) 16 (23.2) 20 (29.0) 31 (44.9)

Prevalence 关n (%)兴b

5.5 ⫾ 0.98 3.5 ⫾ 0.98 2.0 (NA)

7.0 ⫾ 0.83 5.3 ⫾ 1.34 5.4 ⫾ 1.67 6.1 ⫾ 1.03d 6.0 ⫾ 1.56d 3.7 ⫾ 1.34d

Colony count (mean ⫾ SD)c

13 (18.8) 5 (7.2) 1 (1.4)

69 (100.0) 5 (7.2) 13 (18.8) 13 (18.8) 23 (33.3) 26 (37.7)

Prevalence 关n (%)兴b

3.9 ⫾ 1.12 3.2 ⫾ 0.99 2.3 (NA)

7.1 ⫾ 1.06 3.0 ⫾ 0.59 3.2 ⫾ 1.14 4.0 ⫾ 1.16 6.1 ⫾ 1.31 3.6 ⫾ 1.46

Colony count (mean ⫾ SD)c

TABLE 2. Prevalences and descriptive statistics of colony counts in the per-protocol population

Prevalence 关n (%)兴b

7.2 ⫾ 1.15 2.4 ⫾ 0.50 4.2 ⫾ 1.94 2.9 ⫾ 0.56 5.3 ⫾ 1.87 2.7 ⫾ 0.80

19 (27.5) 14 (20.3) 1 (1.4)

Postmenstrual

Colony count (mean ⫾ SD)c

69 (100.0) 8 (11.6) 8 (11.6) 8 (11.6) 15 (21.7) 16 (23.2)

4.0 ⫾ 1.34 4.1 ⫾ 0.96 3.2 ⫾ 0.76

Midmenstrual

Prevalence 关n (%)兴b

7.1 ⫾ 0.93 2.6 ⫾ 0.42 4.0 ⫾ 2.05 4.0 ⫾ 1.70 6.0 ⫾ 1.82 4.0 ⫾ 1.75

15 (21.7) 11 (15.9) 2 (2.9)

Reference tampon (n ⫽ 69)

Colony count (mean ⫾ SD)c

69 (100.0) 8 (11.6) 15 (21.7) 11 (15.9) 24 (34.8) 20 (29.0)

4.2 ⫾ 1.59 2.6 ⫾ 0.76 NA

Premenstruala

Prevalence 关n (%)兴b

6.8 ⫾ 0.75 5.1 ⫾ 1.51 4.2 ⫾ 1.58 5.6 ⫾ 1.37 5.7 ⫾ 1.42 3.5 ⫾ 1.23

16 (23.2) 8 (11.6) 0 (0.0)

Postmenstrual

Colony count (mean ⫾ SD)c

69 (100.0) 18 (26.1) 27 (39.1) 19 (27.5) 20 (29.0) 31 (44.9)

4.4 ⫾ 1.19 3.2 ⫾ 1.03d NA

Midmenstrual

Prevalence 关n (%)兴b

6.9 ⫾ 1.27 3.0 ⫾ 0.73 2.7 ⫾ 1.03 4.3 ⫾ 1.53 5.6 ⫾ 1.15 3.2 ⫾ 1.12

14 (20.3) 10 (14.5) 0 (0.0)

Test tampon (n ⫽ 69)

69 (100.0) 4 (5.8) 10 (14.5) 9 (13.0) 12 (17.4) 14 (20.3)

3.4 ⫾ 0.92 4.3 ⫾ 0.54 2.6 (NA)

Premenstruala

14 (20.3) 7 (10.1) 1 (1.4)

a The number of subjects at the premenstrual visit was too small for statistical testing for S. aureus (test and reference tampons) and Candida species (test and reference tampons). b Prevalences are given as the absolute number of subjects and as a percentage of the per-protocol population. Mean colony counts expressed as log10 CFU/ml of vaginal secretion. Means are geometric means. NA, not applicable. P ⬍ 0.05 for the difference within test or reference tampons compared to each corresponding premenstrual time point.

c

d

croorganisms in the vaginal microflora differed from the preand postmenstrual observations for a variety of organisms in subjects using either the test or reference tampons. Regardless of which tampon was used, midmenstrual prevalence did not change or increased only slightly relative to premenstrual prevalence for Enterococcus species, C. albicans, and other Candida species, whereas it was increased more notably for S. aureus, E. coli, group B Streptococcus, G. vaginalis, and anaerobic Gramnegative rods. None of these changes in prevalence was statistically significant for either cycle of tampon use. In both cycles of tampon use, for both the test and the reference tampons, mean colony counts of all microbes, except Lactobacillus species and C. albicans, increased slightly from pre- to midmenstrual samples. Only isolated changes in colony counts were statistically significant, however. During test tampon use, colony counts for C. albicans were significantly reduced, while during reference tampon use, counts for group B Streptococcus, G. vaginalis, and anaerobic Gram-negative rods were significantly increased. None of these changes was considered to be of clinical significance, and none was accompanied by clinical signs or symptoms. Differences between the pre- and postmenstrual visits were not found to be statistically significant for any microorganism. In several cases, statistical comparisons of changes in colony counts could not be conducted due to the small number of subjects affected. Lactobacillus was present in the vaginal microflora of nearly all of the subjects in the perprotocol population throughout the course of the study. Lactobacillus was present in the premenstrual, midmenstrual, and postmenstrual vaginal microflora of all 69 subjects (100%) when they were using the test tampon. Similarly, the microbe was present premenstrually and postmenstrually in all 69 subjects (100%) and midmenstrually in 68 subjects (98.6%) when they were using the reference tampon. Thus, the prevalence of this microorganism did not change throughout the study for either of the two study products. Overall, the colony counts for Lactobacillus species throughout the study remained stable, with mean values ranging from 6.8 to 7.2 log10 CFU/ml. The slight changes in the colony counts at the midmenstrual and postmenstrual visits compared with the premenstrual visit were not statistically significant in the cycles when either the test tampon or the reference tampon was used. Differences between tampons in vaginal microflora. No statistically significant differences between test and reference tampons were found in prevalence or colony counts at any visit or in changes in prevalence or colony counts between the premenstrual and the midmenstrual or postmenstrual visits for any microorganism evaluated. Incidence. For most organisms, there were a few new occurrences observed at the midmenstrual visit. The highest such incidences were seen for S. aureus, E. coli, and anaerobic Gram-negative rods, with ⱖ17 subjects with new occurrences at the midmenstrual visit compared to the premenstrual visit in both cycles of tampon use. There were no statistically significant differences between the cycles of test tampon use versus reference tampon use. Colposcopy and vaginal pH results. No abnormal findings after use of either test or reference tampons were found on colposcopic examinations in either the per-protocol or ITT populations (data not shown). Vaginal/cervical irritation, inflammation, infection, or dryness did not occur with either

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CHASE ET AL. TABLE 3. Vaginal pH for the per-protocol population Vaginal pH Test tampon (n ⫽ 69)

Parameter

Mean ⫾ SD Minimum 25% quartile Median 75% quartile Maximum

Reference tampon (n ⫽ 69)

Premenstrual visit

Postmenstrual visit

Difference between visits

Premenstrual visit

Postmenstrual visit

Difference between visits

4.0 ⫾ 0.29 3.6 3.6 4.1 4.1 4.4

4.0 ⫾ 0.35 3.6 3.6 4.1 4.1 4.7

0.0 ⫾ 0.40 ⫺0.8 ⫺0.3 0.0 0.5 1.1

3.9 ⫾ 0.28 3.6 3.6 4.1 4.1 4.4

4.0 ⫾ 0.33 3.6 3.6 4.1 4.1 5.0

0.1 ⫾ 0.36 ⫺0.5 0.0 0.0 0.3 0.9

study product. Vaginal pH values were virtually identical at the postmenstrual visits compared with the premenstrual visits, regardless of which tampon was used during the menstrual period (Table 3). The mean change in vaginal pH between the premenstrual and postmenstrual visits was 0.0 ⫾ 0.40 (n ⫽ 69) during the use of test tampons versus 0.1 ⫾ 0.36 (n ⫽ 69) during the use of reference tampons. Summary of tampon use. Diaries kept by subjects demonstrated no clinically important differences in the details of tampon use either between those wearing the test or reference tampon or between subjects randomized to the two treatment sequences (Table 4). A single tampon was used for a mean of approximately 5.5 h for both cycles and both tampons. The average maximum total time of tampon use was approximately 97 h. In general, women used between 10 and 39 tampons per period. Overall, the mean and median number of tampons used per period was 18, with the range being 10 to 39. Although not recommended, several subjects used a tampon longer than 8 h. The number of tampons used longer than 8 h (85 and 82 tampons in the test and reference tampon groups, respectively) and the mean duration of use were very similar between both cycles of tampon use. Adverse events. Of the 80 subjects included in the ITT population, 14/80 (17.5%) reported 16 adverse events when using the test tampon and 11/80 (13.8%) reported 12 such events when using the reference tampon. No adverse event was judged by the investigators to be related to a study tampon. Overall, adverse events occurring with the highest frequency were nervous system disorders (13 subjects; 16.3%) followed by respiratory disorders (4 subjects; 5.0%). Fewer than 4% of subjects had adverse events affecting any other system organ class. Headache, reported in 11/80 subjects (13.8%), was by far the most common adverse event; however, the incidence of

TABLE 4. Summary of tampon use per subject in the intent-to-treat population Result for: Parameter

Test tampon (n ⫽ 80)

Reference tampon (n ⫽ 80)

Avg time of single tampon use in h:min (mean ⫾ SD) Total time of tampon use/period in h:min (mean ⫾ SD) No. of tampons used/cycle (mean ⫾ SD)

5:24 ⫾ 0:56

5:23 ⫾ 0:54

96:33 ⫾ 18:02

96:59 ⫾ 19:25

18.3 ⫾ 4.13

18.5 ⫾ 4.26

headache did not differ between study products (6 subjects; 7.5% with each product). No difference in the incidence or type of adverse events between period of use of the test and reference tampons was noted. Serious adverse events did not occur in the course of the study. Only minor changes of no clinical relevance were noted in blood pressure, heart rate, and oral temperature throughout the study in both the ITT and the per-protocol populations. No between-tampon differences in vital signs were apparent. DISCUSSION Changes in vaginal microflora—both qualitative and quantitative—through the menstrual cycle in this study were similar between the cycles of use of the two different tampons and similar to those reported from earlier studies of tampons and other catamenial products (2, 7–11, 15, 16, 18). In particular, the results of this study are very similar to the results of our previous study of the original apertured film-covered tampon compared to the then current fleece-covered tampon (1). Thus, the prevalence and mean colony counts of anaerobic Gramnegative rods, as well as G. vaginalis, Enterococcus spp., group B Streptococcus, E. coli, and S. aureus, generally increased numerically if not statistically from the pre- to midmenstrual visits, and these values returned or started to return to the premenstrual values by the postmenstrual visit. Unlike our previous study, the mean colony counts of C. albicans decreased somewhat from pre- to midmenstrual samples in both cycles; the difference was statistically significant during the cycle of test tampon use but not during the cycle of reference tampon use. Neither of the two tampons was associated with clinically significant changes in any of the microorganisms evaluated during the menstrual cycle in which it was used. This point is particularly important with respect to S. aureus because of its association with menstrual toxic shock syndrome (TSS). During menstrual use of either the test or reference tampons, counts of S. aureus increased to a mean of only about 105 CFU/ml, which is several orders of magnitude lower than the density at postexponential phase, which is when TSS toxin 1 (TSST-1) is produced (12). Study tampons were not evaluated for microbial content after use in this study, but in vitro studies (P. M. Schlievert, unpublished data) have revealed no untoward effects of the test or reference tampon (or their component materials) on microbial growth or activity (e.g., S. aureus growth or TSST-1 production). Previous in vivo studies found that the microbial

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content of tampons was qualitatively similar to, but perhaps quantitatively less than, that of the vagina (e.g., see references 9 and 10). The apparent lack of change in either prevalence or colony counts of Lactobacillus species in the current study is noteworthy. Every subject had lactobacilli in both pre- and postmenstrual samples from both cycles, only one of these subjects lost lactobacilli during menses (during use of the reference tampon), and in all other subjects, the mean colony counts of lactobacilli remained consistently at the same high levels throughout the cycles (approximately 107 CFU/ml), even during the midmenstrual visit. These findings are especially noteworthy because (i) lactobacilli are the predominant microbes in the healthy vagina and are considered to be responsible for preventing overgrowth of potentially pathogenic microbes and (ii) previous studies reported declines in prevalence and/or abundance of lactobacilli during menses. Eschenbach et al. (3) found that the proportion of women with semiquantitatively high levels of Lactobacillus increased from 70% on days 1 to 5 of the cycle to 92% on days 7 to 12. When vaginal swabs were taken from women using tampons during their menstrual periods, Lactobacillus prevalence and colony counts were shown to increase from day 2 to day 4 to day 21: from 78% to 84% to 91% and from 6.21 to 7.08 to 7.91 log10 CFU/ml, respectively (9). In our previous study (1), about 10% of the subjects lost Lactobacillus during menses (while using either the test or reference tampons), and among those who retained Lactobacillus, there was an average reduction in colony counts of about a half log. There is not an obvious explanation for the differences among these studies with respect to the effect of menses on lactobacilli, especially for the difference between the present study and our previous study (1), in which the reference tampons differed only in the shape of the grooves pressed into the tampon (straight in the previous study, curved in the present study). Differences between our two studies (conducted in Germany using the same laboratory for microbiological evaluations) and earlier studies (conducted in the United States using different laboratories (3, 8–11) could reflect differences in study populations, laboratory methods, or tampons used in the studies. One also might speculate that, in the time span covered by these different studies, there has been a subtle shift in the lactobacilli colonizing the human vagina to forms that are more tolerant of the conditions during menses and/or more resistant to the effects of other microbes whose abundance increases during menses. It also is possible that slight changes in tampon materials (e.g., finishes used on absorbent fibers or cover materials) have resulted in the tampons having positive effects on lactobacilli, such as stabilizing them in the vaginal ecosystem during menses. Further studies would be required to evaluate these possibilities. As in our previous study (1), visual pelvic exams found no signs of vaginal or cervical irritation or ulcerations either during or after use of either the test or the reference tampon, and colposcopic examinations showed no evidence of microulcerations after the use of either tampon. Diary records demonstrated that both test and reference tampons were well tolerated. The majority of adverse events were of mild or moderate intensity and unrelated to the study tampons. No serious adverse events were reported. The average tampon wear times were similar in both treatment se-

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quences and were comparable to that reported in our previous study of the original film-covered tampon (1) and in another fairly recent study of a new tampon (14). In conclusion, the findings of this study demonstrate that the new tampon with the winged apertured film cover has no adverse effects on the vaginal microflora or on the vaginal and cervical epithelium and is as safe and well tolerated as the current commercially marketed tampon with a nonwoven fleece cover. In comparing the results of this study to those of our safety study of the original tampon with a film cover without wings (1), we can conclude that improving the tampon’s leakage performance with the four-wing design does not affect the safety or tolerability of the tampon. ACKNOWLEDGMENTS The authors and their companies are especially indebted to the members of the Tampon Study Group for their dedicated efforts in conducting this study: Petra Weissenberger, the principal investigator, and her colleagues at Quintiles GmbH, Neu Isenburg, Germany; and the clinical investigators, Wolfram Brach, Dietzenbach, Germany, Peter Rosenkranz, Langen, Germany, and Peter Schwaner, Frankfurt, Germany. We also thank Patrick M. Schlievert, University of Minnesota, Minneapolis, and Sharon L. Hillier, University of Pittsburgh, Pittsburgh, PA, for advice on the design of the study and for reviewing the data. We also are grateful to Diann Glickman and Jane Murphy, Zola Associates, for their assistance in preparing the manuscript. This study was funded by Johnson & Johnson Consumer Companies, Inc. David Chase and Berenike Schenkel are employees of Johnson & Johnson Consumer Companies, Inc., and Johnson & Johnson GmbH, respectively. David Chase is listed as a coinventor on patent applications covering the test tampon used in this study; as an employee of Johnson & Johnson Consumer Companies, Inc., he will not receive royalties if these patents are issued. Anne-Marie Fahr and Ulrich Eigner are employees of Laboratory Limbach, which was contracted by Johnson & Johnson GmbH and Johnson & Johnson Consumer & Personal Products Worldwide to perform the microbiological evaluations in this study; they hold no other financial interest in Johnson & Johnson. REFERENCES 1. Chase, D. J., B. P. Schenkel, A. M. Fahr, U. Eigner, and the Tampon Study Group. 2007. A prospective, randomized, double-blind study of vaginal microflora and epithelium in women using a tampon with an apertured film cover compared with those in women using a commercial tampon with a cover of nonwoven fleece. J. Clin. Microbiol. 45:1219–1224. 2. Chow, A. W., and K. H. Bartlett. 1989. Sequential assessment of vaginal microflora in healthy women randomly assigned to tampon or napkin use. Rev. Infect. Dis. 11:S68–S73. 3. Eschenbach, D. A., S. S. Thwin, D. L. Patton, T. M. Hooton, A. E. Stapleton, K. Agnew, C. Winter, A. Meier, and W. E. Stamm. 2000. Influence of the normal menstrual cycle on vaginal tissue, discharge, and microflora. Clin. Infect. Dis. 30:901–907. 4. European Commission on Consumer Affairs. 2002. General product safety directive. http://ec.europa.eu/comm/consumers/cons_safe/prod_safe/ gpsd/currentGPSD_en.htm. 5. European Disposables and Nonwovens Association (EDANA). 1999. Code of practice for tampons, version 1. http://www.edana.org. 6. Hochwalt, J. M., F. C. Sarbaugh, and J. D. Lucas. 2001. Clinical safety of a layered-fiber tampon. Obstet. Gynecol. 97:19S–20S. 7. Morris, C. A., and D. F. Morris. 1967. Normal vaginal microbiology of women of childbearing age in relation to the use of oral contraceptives and vaginal tampons. J. Clin. Pathol. 20:636–640. 8. Onderdonk, A. B. 1998. Assessment of vaginal microflora during use of various catamenial products, p. 16–17. In J. Arbuthnott and B. Furman (ed.), European Conference on Toxic Shock Syndrome. Royal Society of Medicine Press Limited, London, England. 9. Onderdonk, A. B., G. R. Zamarchi, M. R. Rodriguez, M. L. Hirsch, A. Munoz, and E. H. Kass. 1987. Qualitative assessment of vaginal microflora during use of tampons of various compositions, Appl. Environ. Microbiol. 53:2779–2784.

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