Efficacy of orally applied probiotic capsules for ... - OptiBac Probiotics

#1487 European Journal of Obstetrics & Gynecology and Reproductive Biology 168 (2013) 75–79

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Efficacy of orally applied probiotic capsules for bacterial vaginosis and other vaginal infections: a double-blind, randomized, placebo-controlled study a b

w O w p .o tiB pt a ib c ac P pr ro ob bio io tic tic s s. co .u k

Goran Vujic a,*, Alenka Jajac Knez b, Vedrana Despot Stefanovic b, Vedrana Kuzmic Vrbanovic b Department of Gynecology and Obstetrics, Medical School, University of Zagreb, Zagreb, Croatia Jadran Galenic Laboratory, 51000 Rijeka, Croatia

A R T I C L E I N F O

A B S T R A C T

Article history: Received 16 February 2012 Received in revised form 30 September 2012 Accepted 31 December 2012

Objective: To assess the efficacy of orally administered capsules containing the probiotics Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14 (Lactogyn, JGL, Rijeka, Croatia) compared to placebo in otherwise healthy women diagnosed with bacterial vaginosis. Study design: Randomized, double-blind, multicentric, placebo-controlled trial, including a total of 544 subjects. Included were women older than 18 years of age, diagnosed with vaginal infection. Subjects received either probiotic (395 subjects or 72.6%) or identical-looking placebo capsules (149 subjects or 27.4%,) per day over a period of 6 weeks. Six and 12 weeks after the beginning of the study, subjects underwent two additional gynecological examinations and their vaginal swabs were evaluated by a clinical cytologist. Results: Mean follow-up period after the baseline visit was 44 days. After this period, restitution to balanced vaginal microbiota was reported in 40 subjects (26.9%) in the placebo group, compared to 243 subjects (61.5%) in the probiotic group. Differences between groups were statistically significant at p < 0.001. After the additional 6 weeks of follow up, normal vaginal microbiota were still present in more than half (51.1%) of subjects in the probiotic group, but only in around one-fifth (20.8%) of subjects who were taking placebo (p < 0.001). Conclusion: Oral probiotics could be an alternative, side effect-free treatment for one of the most common indications in gynecology, combining the good aspects of both metronidazole and vaginal capsules. ß 2013 Elsevier Ireland Ltd. All rights reserved.

Keywords: Bacterial vaginosis Oral probiotics Placebo-controlled study

1. Introduction

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Bacterial vaginosis is a very common vaginal infection, with a prevalence of 20% in women of reproductive age [1]. Although bacterial vaginosis is mostly a mild and in some cases even asymptomatic disease, it can nevertheless be a cause of endometritis and pelvic inflammatory disease [2,3]. Complications in pregnancy, such as miscarriage, chorioamnionitis, premature rupture of membranes, preterm labor and postpartal endometritis, can all result from bacterial vaginosis [4,5]. In women undergoing in vitro fertilization, bacterial vaginosis reduces the percentage of implanted gametes and can also be the cause of early pregnancy loss [6,7]. The basic pathological process in this disease is the overgrowth of anaerobic microorganisms over the normal vaginal microbiota. These pathogens include Gardnerella vaginalis and sometimes Mycoplasma hominis and Mobyluncus species. It leads to an increase of vaginal pH value and the onset of unpleasantly

? Corresponding author. Tel.: +385 1 460 46 46. E-mail address: [email protected] (G. Vujic). 0301-2115/$ – see front matter ß 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ejogrb.2012.12.031

smelling vaginal discharge. Healthy vaginal microbiota include mostly microorganisms from Lactobacillus species, whose role is to prevent colonization by the aforementioned pathogens. Lactobacilli produce lactic acid, thus decreasing the vaginal pH [8] and hydrogen peroxide, which is toxic to other microorganisms and prevents their colonization in the vagina [9,10]. Bacterial vaginosis is most often treated with metronidazole, which has no effect on lactobacilli but acts against anaerobic microorganisms. Clindamycin is also routinely used [11,12]. Although these are considered to be safe drugs, there are a number of side-effects noted with their use. With metronidazole, gastrointestinal disturbances such as nausea and vomiting, and less frequently, metallic taste in mouth, paresthesias and leucopenia have been reported [13]. The most common sideeffects of clindamycin use are sensitivity reactions manifesting as rash, and gastrointestinal symptoms such as anorexia, abdominal pain, nausea, vomiting and diarrhea. The latter can also be a symptom of pseudomembraneous enterocolitis. Less frequent side-effects include neutropenia, leucopenia, jaundice and skeletal muscle paralysis [14]. Both drugs can be used orally as well as vaginally. Ten to 15% of patients do not respond to the initial

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G. Vujic et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 168 (2013) 75–79

rhamnosus GR-1 and L. reuteri RC-14) or two identical-looking placebo capsules per day over the period of 6 weeks. Six and 12 weeks after the beginning of the study, all subjects underwent two additional gynecological examinations. In subjects with bacterial vaginosis their vaginal smears were evaluated following the procedure used at inclusion. In cases of vulvovaginal candidiasis, evaluation was done by determination of vaginal pH and vaginal discharge wet mount analysis with 10% KOH, while all subjects with trichomoniasis received a single oral dose of 2 grams of metronidazole. Additionally, vaginal swabs from all subjects were taken for evaluation by a clinical cytologist as well as Lactobacillus cultivation (recovery) on agar plates. Lactobacillus recovery from the vagina was done following the method described by Anukam et al. [24]. After dislodging the individual cells in sterile PBS, dilutions were plated on agar plates with or without tetracycline (50 mg/ml). After incubation, plates were analyzed visually, Gram stained and tested for catalase reaction. Following such procedure, pale-yellow, tetracyclineresistant, Gram-positive and catalase-negative colonies were considered to belong to Lactobacillus sp. The primary outcome of the study was defined as the rate of restitution of the normal vaginal microbiota after the follow-up period of 6 weeks. Furthermore, we observed the rate of recurrence of vaginosis after the additional 6 weeks during which period subjects did not take the study medication. Sample size was calculated so that sufficient statistical power (b 0.90) with appropriately low probability of type-1 error (a < 0.05) could be achieved. We used NCSS PASS software [25], with the null hypothesis of achieving a 40% cure rate in placebo group vs. the 65% cure rate in the probiotic group. The null hypothesis was based on our previous experience with this probiotic preparation (personal communication). Such assumption resulted in a sample size of 120 subjects per study group. In the end, however, a much larger sample of 544 women was included in the study. All statistical analyses were performed with Statistica software (version 9, StatSoft, Inc, Tulsa, OK, USA). Rates and proportions were calculated for categorical data and medians and ranges for continuous data. Differences in continuous variables were analyzed by means of Student’s t-test (normally distributed values) or Mann–Whitney U test (non-normally distributed data). Normality of distribution was tested by the Shapiro–Wilk test. For categorical variables, differences were analyzed by means of the chi-square test. All reported p values are two sided. A p value of 109 CFU Lactobacillus

3. Results

During the 12 month period from March 2008 to March 2009, a total number of 544 subjects were included in the study. For a detailed inclusion flow chart please see Fig. 1. Study subjects were randomized to two study groups: placebo (149 subjects or 27.39%) and probiotic (395 subjects or 72.61%). After securing that the necessary minimum of subjects was included in the placebo group, increasing the number of subjects in the probiotic group was the most efficient way to achieve desired statistical power, without denying treatment to subjects affected by bacterial vaginosis. At the baseline visit, subjects in the two study groups were comparable in terms of demographic data – age, number of births, number of sexual partners, age at the first sexual intercourse, habits (smoking and alcohol consumption), marital status and contraception method used (Table 1). Additionally, cytologic smear findings were comparable in both study groups at the baseline visit (p = 0.149; Table 2). The mean follow-up period after the baseline visit was 44.16 days, i.e. approximately 6 weeks and 2 days, and was not significantly different between the two groups (p = 0.733). During


Enrollment

77

Assessed for eligibility (n=743)

Excluded (n=92) Not meeting inclusion criteria (n=27) Declined to participate (n=63) Other reasons (n=2)

Randomized (n=651)


Allocaon

Allocated to intervention (n=466)

Allocated to placebo (n=185)

Follow -Up

Lost to follow-up (n=22) Non-compliant or stopped treatment completely (n=11) Not meeting inclusion criteria (38)

Lost to follow-up (n=15) Non-compliant or stopped treatment completely (n=9) Not meeting inclusion criteria (12)

Analysis

Analysed (n=149)

Analysed (n=395)

Fig. 1. Flow diagram.

Table 1 Baseline characteristics of patients. Characteristic

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Age (range) Births Number of sexual partners 1–3 4–6 >6 Age at the first intercourse 20 years Smoking (cigarettes/day) None 20 Alcohol consumption None Occasionally Daily Marital status Married Never married Separated or divorced Contraception method Condom Oral contraceptives IUD Natural/other

Placebo group (N = 149)

Probiotic group (N = 395)

p value

32.19 (18–54) 1.81

33.26 (18–58) 1.73

0.234 0.463

74.50% 18.79% 6.71%

70.89% 18.73% 10.38%

0.438

6.04% 79.87% 14.09%

4.56% 74.43% 21.01%

0.162

68.46% 12.75% 14.77% 4.03%

64.56% 16.46% 13.92% 5.06%

0.680

56.38% 40.94% 2.68%

50.07% 47.65% 2.28%

0.583

63.09% 32.89% 4.04%

56.20% 36.96% 6.84%

0.248

16.78% 24.83% 8.05% 50.33%

24.81% 20.51% 8.10% 46.48%

0.224

the follow-up period, subjects were taking either placebo or the probiotic capsules twice a day, as described earlier. Compliance was comparable between the two study groups (97.64% in the placebo vs. 91.67% in the probiotic group, p = 0.128). After the above-mentioned follow-up period, restitution to balanced vaginal microbiota was reported in 40 subjects (26.85%) in the placebo group, compared to 243 subjects (61.52%) in the probiotic group. Differences between groups were statistically significant at p < 0.001. At this time point, the number needed to treat (NNT) was 2.9 with a relative risk reduction (RRR) of 47.4%. After the additional 6 weeks of follow-up, normal vaginal microbiota were still present in more than half (51.14%) of subjects in the probiotic group, but only in around one-fifth (20.81%) of subjects who were taking placebo (p < 0.001). At this later time point the NNT was 3.3, while the RRR was 38.3%. Detailed results of cytologic smear findings are laid out in Table 3. All subjects with

Table 2 Cytologic smear results at baseline. Finding

Gardnerella vag. Fungi Mixed flora Trichomonas vag. Gardnerella vag. + mycosis Total

Placebo group

Probiotic group

N

%

N

%

31 50 49 2 17 149

20.81 33.56 32.89 1.34 11.41 100.00

78 107 172 8 30 395

19.75 27.09 43.54 2.03 7.59 100.00

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Table 3 Cytologic smear results after 6 and 12 weeks of follow-up. Finding

Baseline + 6 weeks Placebo group

Gardnerella vag. Fungi Mixed flora Trichomonas vag. Bacillus vag. Gardnerella vag. + mycosis Total

Baseline + 12 weeks Probiotic group

Probiotic group

%

N

%

N

%

N

%

11 14 83 0 40 1 149

7.38 9.40 55.70 0.00 26.85 0.67 100.00

12 27 109 1 243 3 395

3.04 6.84 27.59 0.25 61.52 0.76 100.00

13 19 84 0 31 2 149

8.72 12.75 56.38 0.00 20.81 1.34 100.00

11 24 155 0 202 3 395

2.78 6.08 39.24 0.00 51.14 0.76 100.00

metronidazole gel in treatment of bacterial vaginosis [30], with comparable results. Similar results were published by Martinez et al. [31] (combination of tinidazole and probiotic in comparison with tinidazole and placebo), and by Ya et al. [28] who recently conducted a randomized, double-blind, placebo-controlled study testing the effectiveness of probiotic vaginal capsules in preventing recurrences of bacterial vaginosis. Although not without certain shortcomings in terms of population size, inclusion conditions or follow up methodology, the above-mentioned studies established a strong case for the existence of beneficiary effect of Lactobacilli in subjects with bacterial vaginosis. With that in mind, we designed the present study to include a relatively large number of subjects from numerous primary care gynecological practitioners. Additionally, we established a scrupulous procedure for both baseline and follow-up diagnostic procedures and closely monitored subject compliance. By applying such a study design we were able to accrue a sufficient number of subjects within a reasonable time frame and avoid errors commonly seen in single-centre trials [32]. Finally, recruiting subjects from a wider population and testing the effects of the treatment in a broader range of clinical settings presented an experimental situation more typical of future use. The main limitation of our study was a relatively short follow-up period of 6 and 12 weeks post-baseline, which was necessary to avoid dispersal of study subjects. Additionally, DNA probes specific for Lactobacillus GR-1 and RC-14 would facilitate identification of strains in the follow-up vaginal swabs, but were unavailable at the time the study was performed. Our findings confirm the positive effects of probiotics described in previously mentioned studies. The results also support the association between the proportion of subjects with positive posttreatment Lactobacillus sp. culture and the proportion of subjects achieving restitution to balanced vaginal microbiota, which was the main outcome of the study. In our opinion, the most promising result of the probiotic therapy as a whole (either as vaginal capsules or as oral therapy) is the potential to reduce or make unnecessary the use of metronidazole in treatment of bacterial vaginosis. Probiotic vaginal capsules have already been described to be more effective than metronidazole for this condition [33], while our results confirm that this is true for the oral probiotic therapy as well. We feel that oral probiotics could be an alternative, side effect-free treatment for a very common disease in gynecological practice, combining the good aspects of both metronidazole and vaginal capsules. We agree with Ya et al. [28] that potential benefit of probiotics versus antibiotics can be confirmed only by means of further randomized clinical trials.


unfavorable results at the second (12 week) follow-up visit received appropriate treatment after their participation in the study was completed. Additional cultivation on agar plates was performed to recover and identify Lactobacilli from the vaginal swabs. High counts (>105 CFU/ml) of lactobacilli were recovered from 81.5% of subjects who received probiotic, compared to only 28.9% of subjects receiving placebo. Thus, restitution to normal (balanced) vaginal microbiota coincided highly (p < 0.001) with presence of lactobacilli in the vaginal swabs on both post-baseline visits. None of the demographic variables described in Table 1 was related significantly to the outcome – the proportion of subjects with the restituted vaginal microbiota after either 6 or 12 weeks post-baseline. Borderline significance (p = 0.082) was found, however, when number of children was compared to the outcome. Women with 3 or more deliveries were less likely to see restitution to normal microbiota, compared to women with 2, 1 or no births. This finding is clearly not due to significantly higher mean age of women with more children (40.1 years for women with 3 or more children as opposed to 29.9 years for women with 2 children or less), because age had no effect on the outcome, as mentioned before. One possible explanation could be that both occurrence and recurrence of bacterial vaginosis (and other female reproductive tract infections) are more common in women with more deliveries, a fact already shown by other researchers [26]. A much larger study with longer follow-up period would be needed, however, to confirm or reject this hypothesis. 4. Comments

Placebo group

N

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Bacterial vaginosis is a condition known to be associated with low numbers of Lactobacillus sp. bacteria in vagina [27]. Although the source of vaginosis (reinfection or relapse) has not been fully elucidated, it is thought that lactobacilli, by means of their production of lactic acid, inhibit growth of other microorganisms, thus inhibiting their overgrowth [20,28]. The same mechanism is responsible for maintaining low vaginal pH values. The most frequently used strains of lactobacilli in therapy are a combination of L. rhamnosus GR-1 and Lactobacillus fermentum RC-14, shown by in vitro studies to posses the ability of adhesion to the cells of vaginal epithelium [29]. Because of this they are able to colonize the vagina after oral application [13]. Studies of the effects of probiotics on bacterial vaginosis are relatively scarce. Anukam et al. [24] conducted a randomized, controlled, double-blind study which confirmed the positive effects of combined use of metronidazole with capsules containing probiotic in comparison to placebo in treatment of bacterial vaginosis. After the 30-day period, 88% of patients who received probiotic had no signs of bacterial vaginosis, in comparison to 40% vaginosis-free patients in placebo group (p < 0.001). The same authors also conducted another randomized clinical trial which compared the efficacy of probiotic vaginal capsules used with the

Condensation Oral probiotics could be an alternative, side effect-free treatment for bacterial vaginosis, combining the good aspects of both metronidazole and vaginal capsules.


References [18]

[19] [20] [21]

[22]

[23] [24]

http://www.who.int/foodsafety/publications/fs_management/en/probiotics.pdf [accessed 11.02.08]. Morelli L, Zonenenschain D, Del Piano M, Cognein P. Utilization of the intestinal tract as a delivery system for urogenital probiotics. Journal of Clinical Gastroenterology 2004;38:S107–10. Reid G, Burton J. Use of lactobacillus to prevent infection by pathogenic bacteria. Microbes and Infection 2002;4:319–24. Falagas ME, Betsi GI, Athanasiou S. Probiotics for the treatment of women with bacterial vaginosis. Clinical Microbiology and Infection 2007;13:657–64. Amsel R, Totten PA, Spiegel CA, Chen KC, Eschenbach D, Holmes KK. Nonspecific vaginitis: diagnostic criteria and microbial and epidemiologic associations. American Journal of Medicine 1983;74:14–22. Nugent RP, Krohn MA, Hillier SL. Reliability of diagnosing bacterial vaginosis is improved by a standardized method of Gram stain interpretation. Journal of Clinical Microbiology 1991;29:297–301. Saghaei M. Random allocation software for parallel group randomized trials. BMC Medical Research Methodology 2004;4:26. Anukam K, Osazuwa E, Ahonkhai I, et al. Augmentation of antimicrobial metronidazole therapy of bacterial vaginosis with oral probiotic Lactobacillus rhamnosus GR1 and Lactobacillus reuteri RC14: randomized double blind placebo controlled trial. Microbes and Infection 2006;8:1450–4. Hintze J. PASS 2008. Kaysville, Utah, USA, NCSS, LLC; 2008. www.ncss.com. Zhang XJ, Shen Q, Wang GY, et al. Risk factors for reproductive tract infections among married women in rural areas of Anhui Province, China. European Journal of Obstetrics Gynecology and Reproductive Biology 2009;147: 187–91. Hellberg D, Nilsson S, Mardh PA. The diagnosis of bacterial vaginosis and vaginal flora changes. Archives of Gynecology and Obstetrics 2001;265:11–5. Ya W, Reifer C, Miller LE. Efficacy of vaginal probiotic capsules for recurrent bacterial vaginosis: a double-blind, randomized, placebo-controlled study. American Journal of Obstetrics and Gynecology 2010;203:e1–6. 120. Boris S, Suarez J, Vazquez F, et al. Adherence of human vaginal Lactobacilli to vaginal epithelial cells and interaction with uropathogens. Infection and Immunity 1998;66:1985–9. Anukam K, Osazuwa E, Osemene GI, Ehigiagbe F, Bruce AW, Reid G. Clinical study comparing probiotic Lactobacillus GR-1 and RC-14 with metronidazole vaginal gel to treat symptomatic bacterial vaginosis. Microbes and Infection 2006;8:2772–6. Martinez RC, Franceschini SA, Patta MC, et al. Improved treatment of vulvovaginal candidiasis with fluconazole plus probiotic Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14. Letters in Applied Microbiology 2009;48:269–74. DeLucca P, Raghavarao D. Effect of investigator bias on the significance level of the Wilcoxon rank-sum test. Biostatistics 2000;1:107–11. Oduyebo OO, Anorlu RI, Ogunsola FT. The effects of antimicrobial therapy on bacterial vaginosis in non-pregnant women. Cochrane Database of Systematic Reviews 2009;CD006055.

w


[1] Forsum U, Holst E, Larsson PG, et al. Bacterial vaginosis—a microbiological and immunological enigma. APMIS 2005;113:81–90. [2] Sweet RL. Gynecologic conditions and bacterial vaginosis: implications for the non-pregnant patient. Infectious Diseases in Obstetrics and Gynecology 2000;8:184–90. [3] Ness RB, Hillier SL, Kip KE, et al. Bacterial vaginosis and risk of pelvic inflammatory disease. Obstetrical and Gynecological Survey 2005;60:99–100. [4] Guaschino S, Ricci E, Franch M, et al. Treatment of asymptomatic bacterial vaginosis to prevent pre term delivery: a randomized trial. European Journal of Obstetrics Gynecology and Reproductive Biology 2003;110:149–52. [5] Leitich H, Bodner-Adler B, Brunbauer M, Kaider A, Egarter C, Husslein P. Bacterial vaginosis as a risk factor for pre-term delivery: a meta analysis. American Journal of Obstetrics and Gynecology 2003;189:139–47. [6] Eckert LO, Moore DE, Patton DL, et al. Relationship of vaginal bacteria and inflammation with conception and early pregnancy loss following in-vitro fertilization. Infectious Diseases in Obstetrics and Gynecology 2003;11:11–7. [7] Wilson JD, Ralph SG, Rutherford AJ. Rates of bacterial vaginosis in women undergoing in vitro fertilisation for different types of infertility. BJOG 2002;109:714–7. [8] Hill GB, Eschenbach DA, Holmes KK. Bacteriology of the vagina. Scandinavian Journal of Urology and Nephrology 1984;86(Suppl):23–9. [9] Klebanoff SJ, Hillier SL, Eschenbach DA, Waltersdorph AM. Control of the microbial flora of the vagina by H202 generating Lactobacilli. Journal of Infectious Diseases 1991;164:94–100. [10] Eschenbach DA, Davick PR, Williams BL, et al. Prevalence of hydrogen peroxide-producing Lactobacillus species in normal women and women with bacterial vaginosis. Journal of Clinical Microbiology 1989;27:251–6. [11] Anon.. CDC sexually transmitted disease treatment guidelines 2006. Morbidity and Mortality Weekly Report 2006;55:50–2. [12] Marrazzo JM. Evolving issues in understanding and treating bacterial vaginosis. Expert Review of Anti-Infective Therapy 2004;2:913–22. [13] Hager WD, Rapp RP. Metronidazole. Obstetrics and Gynecology Clinics of North America 1992;19:497–510. [14] Trexler MF, Fraser TG, Jones MP. Fulminant pseudomembranous colitis caused by clindamycin phosphate vaginal cream. American Journal of Gastroenterology 1997;92:2112–3. [15] Bradshaw CS, Morton AN, Hocking J, et al. High recurrence rates of bacterial vaginosis over the course of 12 months after oral metronidazole therapy and factors associated with recurrence. Journal of Infectious Diseases 2006;193:1478–86. [16] Reid G, Bruce AW, Fraser N, et al. Oral probiotics can resolve urogenital infections. FEMS Immunology and Medical Microbiology 2001;30:49–52. [17] Report of a Joint FAO/WHO Expert Consultation on health and nutritional properties of probiotics in food including powder milk with live lactic acid bacteria. American Co´rdoba Park Hotel, Co´rdoba, Argentina, 1–4 October 2001.

[25] [26]

[27]

[28]

[29]

[30]

[31]

[32]

[33]

79