Abstract

Forty-two healthy women were randomized to receive one of three encapsulated Lactobacillus rhamnosus GR-1 plus Lactobacillus fermentum RC-14 probiotic dosage regimens or L. rhamnosus GG by mouth each day for 28 days. However, the vaginal flora, assessed by Nugent scoring, was only normal in 40% of the cases, and 14 patients had asymptomatic bacterial vaginosis. Treatment with L. rhamnosus GR-1/L. fermentum RC-14 once and twice daily correlated with a healthy vaginal flora in up to 90% of patients, and 7/11 patients with bacterial vaginosis converted to normal or intermediate scores within 1 month. Ingestion of L. rhamnosus GG failed to have an effect. This study confirms the potential efficacy of orally administered lactobacilli as a non-chemotherapeutic means to restore and maintain a normal urogenital flora, and shows that over 108 viable organisms per day is the required dose.

Introduction

Recent studies have emphasized the importance of a healthy, lactobacilli dominated flora not only to prevent sexually transmitted diseases [1] and preterm labour [2], but also to maintain the quality of life of women [3]. In an attempt to develop a non-chemotherapeutic means to restore and maintain a healthy urogenital tract, probiotic therapy using lactobacilli has been considered, and there is evidence to indicate that certain strains can be effective when inserted directly into the vagina or when ascending from the rectum after oral ingestion [4–6]. Two strains, Lactobacillus rhamnosus GR-1 and Lactobacillus fermentum RC-14, appear to be particularly adept at the latter [6]. Repeated intake of probiotics could be important not only in women prone to urogenital infections, but also for healthy women as their vaginal flora is often depleted of lactobacilli thereby increasing the risk of infection [7,8].

The present study was designed to determine the dose required for probiotic lactobacilli to impact the vaginal flora, and to compare a commercial product with strains GR-1 and RC-14. A Gram stain system, proven to be effective in assessing the normality or ‘health’ of the vaginal flora without the need for culture [9], was used. L. rhamnosus GG was selected as a control arm of the study, because its ingestion fails to affect the vaginal flora [10].

Methods

Lactobacillus strains

Strains L. rhamnosus GR-1 and L. fermentum RC-14 were grown in MRS broth (Sigma, Detroit, MI, USA), tested for purity, and freeze dried in equal amounts into gelatin capsules in dosage forms of 8×108 and 6×109 viable organisms. Commercially available capsules containing 1010 viable L. rhamnosus GG were purchased. All capsules were placed in vials and stored in a household refrigerator until use. Viable counts were carried out at regular intervals to determine shelf life, and no significant loss of viability was found during the duration of the study.

Subjects and randomization

The subjects, primarily Caucasian, were recruited through family practices in London, ON, Canada. Each subject voluntarily signed an informed consent using a format approved by the Ethics Review Board of the University of Western Ontario. The inclusion criteria were for healthy women, 17 years and over (actual range was 17–50 with mean of 31±8), who were currently free from urogenital infections (urinary tract infection (UTI), bacterial vaginosis (BV), or yeast vaginitis) and were not on long term, low dose antibiotics for UTI. The exclusion criteria were patients with abnormal renal function (serum creatinine >110 µmol l−1, upper limit 90 µmol l−1) or pyelonephritis, women who were pregnant, women who were lactose intolerant or receiving prednisone, immunosuppressive drugs, antimicrobial therapy or were using nonoxynol-9 as a spermicide agent. No subject received antibiotics during the study.

The capsules were dispensed in a randomized manner into vials containing one capsule per day of 1010 of GG (Group 4), 8×108 (Group 1) and 6×109 (Group 3) of GR-1/RC-14, and two per day of the 8×108=1.6×109 (Group 2) GR-1/RC-14 dose. Treatment was given for 28 days. The technician evaluating the Nugent scores was blinded as to the treatments given, as were the researchers and patients, except for those subjects given twice daily therapy.

Sample processing

Deep vaginal swabs were collected within 2 days prior to study, then on days 7, 14, 21, 28, 35, and 41. The swabs were placed onto glass slides and examined using the Nugent scale (0–3 normal; 4–6 intermediate; 7–10 BV) [9].

Results

Compliance was excellent and all 42 subjects completed the study, although four did not provide a final vaginal swab. None of the patients reported symptomatic yeast vaginitis, UTI or BV or adverse side effects during the 6 week test period. While all the subjects reported feeling normal with respect to the urogenital tract upon entry into the study, only 17/42 (40%) actually showed a normal Nugent score. Indeed, asymptomatic BV was diagnosed by Nugent scoring in 4/10 women in Group 1, 3/12 in Group 2, 4/11 in Group 3 and 3/9 in the GG control Group 4 (Tables 1–4). BV scores reverted to normal or intermediate at day 28 in 7/11 (64%) subjects given one of three forms of GR-1/RC-14 therapy. At 2 week follow-up, 2/9 (22.2%) patients in the GG control Group 4 had a BV score, compared to 4/29 (13.8%) in the GR-1/RC-14 treated group (P=0.613 Fisher's Exact test).

Table 1

Nugent scoring outcomes of patients in Group 1 given once daily 8×108L. rhamnosus GR-1 and L. fermentum RC-14

Patient No. History Day 0 Day 7 Day 14 Day 21 Day 28 Day 35 Day 42 
104 YV BV BV BV BV BV BV N/A 
110 YV/UTI Normal Normal Normal Normal Normal Normal Normal 
111 YV Int. Int. Int. Int. Normal Normal Int. 
113 UTI Int. Normal Int. Int. Int. BV Int. 
116 UTI Normal Normal Normal Normal Normal Normal Normal 
117 UTI/YV/BV Normal Normal Int. Normal Normal Normal Normal 
118 YV BV Normal Normal Normal Normal Normal Normal 
120 YV BV Int. Int. Int. Int. Int. BV 
125 YV/UTI Normal Normal Normal Normal Normal Normal Normal 
133 None BV BV BV BV BV BV BV 
Patient No. History Day 0 Day 7 Day 14 Day 21 Day 28 Day 35 Day 42 
104 YV BV BV BV BV BV BV N/A 
110 YV/UTI Normal Normal Normal Normal Normal Normal Normal 
111 YV Int. Int. Int. Int. Normal Normal Int. 
113 UTI Int. Normal Int. Int. Int. BV Int. 
116 UTI Normal Normal Normal Normal Normal Normal Normal 
117 UTI/YV/BV Normal Normal Int. Normal Normal Normal Normal 
118 YV BV Normal Normal Normal Normal Normal Normal 
120 YV BV Int. Int. Int. Int. Int. BV 
125 YV/UTI Normal Normal Normal Normal Normal Normal Normal 
133 None BV BV BV BV BV BV BV 

None, no past history of urogenital infection; BV, asymptomatic bacterial vaginosis and scores within the BV Nugent range of 7–10; YV, yeast vaginitis; UTI, urinary tract infection; Normal, scores within the normal Nugent range of 0–3; Int., scores within the intermediate Nugent range of 4–6; N/A, not available.

Table 2

Nugent scoring outcomes of patients in Group 2 given 8×108L. rhamnosus GR-1 and L. fermentum RC-14 twice daily (that is 1.6×109 daily dose)

Patient No. History Day 0 Day 7 Day 14 Day 21 Day 28 Day 35 Day 42 
101 YV Normal Normal Normal Normal Normal Normal Normal 
102 YV Int. Int. Normal Normal Normal Int. Normal 
103 UTI Int. Normal Normal Int. Normal Normal Normal 
105 YV/UTI BV BV Int. Int. Int. Normal Normal 
106 YV/UTI BV BV Int. Normal Normal Normal Normal 
107 YV/BV Normal Normal Int. N/A Int. Int. Int. 
108 UTI BV BV Int. N/A Int. N/A N/A 
109 YV/UTI Normal Normal Normal Normal Normal Normal Normal 
121 YV Normal Normal Normal Normal Normal Normal Normal 
139 YV Int. Int. Int. Normal Normal Normal Normal 
143 BV/YV Normal Normal Int. Normal Normal Normal N/A 
145 YV Normal Normal Normal Normal Normal Normal Normal 
Patient No. History Day 0 Day 7 Day 14 Day 21 Day 28 Day 35 Day 42 
101 YV Normal Normal Normal Normal Normal Normal Normal 
102 YV Int. Int. Normal Normal Normal Int. Normal 
103 UTI Int. Normal Normal Int. Normal Normal Normal 
105 YV/UTI BV BV Int. Int. Int. Normal Normal 
106 YV/UTI BV BV Int. Normal Normal Normal Normal 
107 YV/BV Normal Normal Int. N/A Int. Int. Int. 
108 UTI BV BV Int. N/A Int. N/A N/A 
109 YV/UTI Normal Normal Normal Normal Normal Normal Normal 
121 YV Normal Normal Normal Normal Normal Normal Normal 
139 YV Int. Int. Int. Normal Normal Normal Normal 
143 BV/YV Normal Normal Int. Normal Normal Normal N/A 
145 YV Normal Normal Normal Normal Normal Normal Normal 

None, no past history of urogenital infection; BV, asymptomatic bacterial vaginosis and scores within the BV Nugent range of 7–10; YV, yeast vaginitis; UTI, urinary tract infection; Normal, scores within the normal Nugent range of 0–3; Int., scores within the intermediate Nugent range of 4–6; N/A, not available.

Table 3

Nugent scoring outcomes of patients in Group 3 given once daily 6×109L. rhamnosus GR-1 and L. fermentum RC-14

Patient No. History Day 0 Day 7 Day 14 Day 21 Day 28 Day 35 Day 42 
114 UTI/YV Normal Normal Normal Normal Normal Normal Normal 
119 UTI/YV Int. Int. Int. Int. Normal Int. Int. 
123 YV Normal Normal Normal Normal Normal N/A Normal 
124 YV BV BV Int. Int. BV BV BV 
126 None Int. BV Int. Int. Int. Int. Int. 
127 YV Int. Int. Int. Int. Int. Int. Int. 
128 UTI/BV/YV BV BV N/A Int. Normal Int. Int. 
129 UTI/BV/YV Normal Normal Normal Normal Normal Normal Int. 
130 UTI BV BV BV Int. Int. Int. Normal 
131 UV Int. Int. Normal Int. Int. Int. Normal 
132 None BV BV BV BV BV BV BV 
Patient No. History Day 0 Day 7 Day 14 Day 21 Day 28 Day 35 Day 42 
114 UTI/YV Normal Normal Normal Normal Normal Normal Normal 
119 UTI/YV Int. Int. Int. Int. Normal Int. Int. 
123 YV Normal Normal Normal Normal Normal N/A Normal 
124 YV BV BV Int. Int. BV BV BV 
126 None Int. BV Int. Int. Int. Int. Int. 
127 YV Int. Int. Int. Int. Int. Int. Int. 
128 UTI/BV/YV BV BV N/A Int. Normal Int. Int. 
129 UTI/BV/YV Normal Normal Normal Normal Normal Normal Int. 
130 UTI BV BV BV Int. Int. Int. Normal 
131 UV Int. Int. Normal Int. Int. Int. Normal 
132 None BV BV BV BV BV BV BV 

None, no past history of urogenital infection; BV, asymptomatic bacterial vaginosis and scores within the BV Nugent range of 7–10; YV, yeast vaginitis; UTI, urinary tract infection; Normal, scores within the normal Nugent range of 0–3; Int., scores within the intermediate Nugent range of 4–6; N/A, not available.

Table 4

Nugent scoring outcomes of patients in Group 4 given once daily 1010L. rhamnosus GG control

Patient# History Day 0 Day 7 Day 14 Day 21 Day 28 Day 35 Day 42 
134 UTI/YV Normal Int. Int. Normal Int. Int. Int. 
135 YV BV BV N/A N/A Int. Int. Int. 
136 BV/YV Normal Normal Normal Normal Normal Normal Normal 
137 UTI/YV Int. Int. Int. Normal Int. Int. BV 
138 YV BV BV BV Normal Normal Normal Normal 
140 UTI/YV Int. Int. Int. Int. Int. Int. Int. 
141 None BV BV BV N/A N/A N/A BV 
142 YV/BV Normal Int. Normal Normal Normal Normal Normal 
144 YV Normal Normal Normal Int. Int. Int. Int. 
Patient# History Day 0 Day 7 Day 14 Day 21 Day 28 Day 35 Day 42 
134 UTI/YV Normal Int. Int. Normal Int. Int. Int. 
135 YV BV BV N/A N/A Int. Int. Int. 
136 BV/YV Normal Normal Normal Normal Normal Normal Normal 
137 UTI/YV Int. Int. Int. Normal Int. Int. BV 
138 YV BV BV BV Normal Normal Normal Normal 
140 UTI/YV Int. Int. Int. Int. Int. Int. Int. 
141 None BV BV BV N/A N/A N/A BV 
142 YV/BV Normal Int. Normal Normal Normal Normal Normal 
144 YV Normal Normal Normal Int. Int. Int. Int. 

None, no past history of urogenital infection; BV, asymptomatic bacterial vaginosis and scores within the BV Nugent range of 7–10; YV, yeast vaginitis; UTI, urinary tract infection; Normal, scores within the normal Nugent range of 0–3; Int., scores within the intermediate Nugent range of 4–6; N/A, not available.

As shown in Table 4 and summarized in Table 5, the control GG group showed no improvement in the number of subjects with normal vaginal flora at the end of 28 days of treatment. On the contrary, treatment with the GR-1/RC-14 combination twice daily (still almost one log less than one dose of the GG control) resulted in 50% more normal scores than before treatment started. Within 2 weeks of completion of treatment, the vaginal flora remained normal (comparing day 0 and day 28) in 90% of women given the twice daily GR-1/RC-14 lactobacilli, and this was significantly better than the GG control (P=0.017).

Table 5

Summary of women with a healthy Nugent score for their vaginal flora before treatment and at days 28 and 42 following treatment with L. rhamnosus GR-1 and L. fermentum RC-14 (Groups 1–3) or L. rhamnosus GG

 Percentage of women with healthy vaginal flora 
 Group 1 (8×108Group 2 (1.6×109Group 3 (6×109Group 4 (1010 GG) 
Before treatment 40 50 27 44 
At end of treatment 60 82 45 38 
Two weeks after treatment 56 90 30 33 
 Percentage of women with healthy vaginal flora 
 Group 1 (8×108Group 2 (1.6×109Group 3 (6×109Group 4 (1010 GG) 
Before treatment 40 50 27 44 
At end of treatment 60 82 45 38 
Two weeks after treatment 56 90 30 33 

As defined by Nugent scoring.

Of the subjects who had a history of yeast vaginitis during the previous 5 years, 7/13 (54%) who did not have a normal flora at entry, developed a normal flora within 28 days of treatment with GR-1/RC-14, while one 1/4 (25%) of the GG controls converted to normal. Of the five subjects who had a history of BV and presented with a BV Nugent score, one (No. 128) converted to a normal flora with GR-1/RC-14 treatment, while none of the GG controls converted. Of subjects with a past history of UTI, treatment with one of the three GR-1/RC-14 doses was better at converting an abnormal flora on day 0 to a normal flora within 28 days (P=0.043).

The conversion to a normal flora within 28 days was significant for women in Group 2 (P=0.017), with a tendency towards significance for Group 3 (P=0.147), and lack of significance for Group 1 (P=0.611 on day 28; P=0.994 on day 42) compared to control Group 4. The dosage forms higher than 8×108 appeared to be necessary for clinical effect.

Discussion

The pool of subjects was quite typical of healthy women in a fairly well educated, middle class community. However, the nature of the study attracted more women with a history of previous urogenital infection than in a random population, and these women likely represent consumers wishing to utilize functional foods as a means of maintaining health [11]. The women were not at high risk of sexually transmitted diseases, unlike some populations studied elsewhere [8], such as those with BV and at greater risk of HIV-1 through sexual intercourse [12]. Their overall history of bladder and vaginal infection was not unexpected, given the high incidence of these diseases amongst women. The low incidence of a history of BV was also not surprising, given its poor diagnosis in community clinics. The prevalence of normal flora at 40% at the start of the study was lower than the 76.5% reported by Schwebke et al. [8], perhaps illustrating the innate abnormality of women with a history of urogenital infections and antibiotic treatment.

The finding that the flora was normal in the majority of subjects after treatment with lactobacilli strains GR-1 and RC-14, and not the control patients taking L. rhamnosus GG, suggested strongly that the two strain combination therapy had an impact on the flora. The ingestion of the GR-1/RC-14 probiotic therapy resulted in the vaginal flora being restored to normal within 28 days in 82% in one group of patients. This is much higher than normal Nugent values reported in a general healthy population of women [8]. Whether or not a longer duration of treatment would mean that the flora could be maintained as normal in all subjects remains to be determined. In subjects who started with a normal flora, 12/13 (92%) retained normality at day 28 and one scored intermediate following GR-1/RC-14 therapy. This is almost double the 48% of women found elsewhere to maintain a normal flora over a menstrual cycle as determined by Nugent scoring [7]. This suggests that daily probiotic use could be considered as a means to maintain a healthy urogenital flora.

The question of how long on therapy did it take to increase the number of normal Nugent scores was not an aim of the study, and while there is an increase in Groups 1 and 2 on day 7 and day 21, no statement of significance can be made.

The dosage forms did not reveal significant differences, and it appears that the range of doses selected for study was too narrow.

There was no significant difference between Group 1 and the control at day 28 or day 42, suggesting that a daily dose greater than 8×108 is required for an effect. The twice daily dose of GR-1/RC-14 contained considerably fewer organisms than those in the GG control product. The results for L. rhamnosus GG are consistent with its inability to colonize the vagina or protect the host from recurrence of UTI [10].

The prevalence of asymptomatic bacterial vaginosis was 26% (11/42) at study commencement as assessed by Nugent evaluation. This is similar to the 20% figure found in a larger study of 635 women [13]. The conversion of BV scores to normal with GR-1/RC-14 therapy (64%) is higher than the spontaneous conversion rate reported at 12% in a previous study [14], implying potential clinical significance with probiotic therapy.

For women prone to recurrence of UTI, GR-1/RC-14 and not GG therapy corresponded with maintenance of a lactobacilli dominated vaginal flora. This reaffirms an earlier finding that prophylactic antibiotics could one day be replaced by oral probiotics as a means to prevent UTI [5].

The study was not designed to determine if the ingested strains were the ones that colonized the vagina. Rather, even if the effect was a result of the therapy creating an environment better able to support indigenous lactobacilli growth [5], the clinical outcome is still important.

In summary, the findings indicate that a daily oral dose of 108 viable probiotic lactobacilli can restore and maintain the urogenital health of women.

Acknowledgements

The assistance of Ms. D. Lam and Drs. J. Hammond, B. Henning, J. Owen, and E. Smith is much appreciated.

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