Sexually Transmitted Diseases:
Probiotics: Potential to Prevent HIV and Sexually Transmitted Infections in Women
Bolton, Michael MD, PhD*; van der Straten, Ariane PhD†; Cohen, Craig R. MD, MPH†
From the *Department of Medicine, Emory University, Atlanta, Georgia; and †Department of Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco, California
Correspondence: Craig R Cohen, MD, MPH, 50 Beale Street, Suite 1200, San Francisco, CA 94117. E-mail: email@example.com.
Received for publication February 2, 2007, and accepted September 12, 2007.
Women are at significant risk of human immunodeficiency virus (HIV) and sexually transmitted infection (STI) acquisition with the genital mucosa serving as the main portal of infection. Exogenously supplied lactobacillus used as a probiotic may prove a cost-effective, female-initiated method to prevent HIV and STI infection in women. A probiotic may act indirectly through treating and preventing recurrent bacterial vaginosis or directly by secreting endogenous (e.g., hydrogen peroxide) and exogenous substances that block HIV and STI transmission. This review summarizes the preclinical and clinical studies that have been conducted so far to test probiotic bacteria for these purposes. Although significant progress has been made in this field, more fundamental research is required to better understand vaginal ecology to maximize probiotic formulations. Once identified, a suitable product will require testing in a well-designed, randomized, placebo-controlled trial to measure its effectiveness in augmenting antibiotic treatment to prevent bacterial vaginosis. If results from such a trial demonstrate efficacy, future studies should be designed to determine whether a probiotic can significantly lower the risk for HIV and STIs in at-risk female populations.
THE HIGH BURDEN OF human immunodeficiency virus (HIV) and sexually transmitted infections (STIs) among women, particularly in developing countries, has drawn attention to developing cost-effective, female-initiated alternatives to the male condom to prevent these infections.1 Probiotics are “live microorganisms, which, when administered in adequate amounts, confer a health benefit on the host.”2 Probiotics could serve as a means to maintain vaginal health and protect against STIs, be genetically modified to inhibit infection by specific sexually transmitted pathogens, and be used in combination with other antimicrobials. This article reviews the current literature and investigations on probiotics for their potential to prevent HIV and STIs in women.
Shortly after birth, the skin, upper respiratory, gastrointestinal, and urogenital tracts become colonized with microorganisms and a lifelong symbiosis begins. The species that occupy the various niches provided by the human body can change based on intrinsic host factors such as stage of life cycle, hormone levels, immune responses, nutritional status, and disease states or can be altered by external factors such as environmental exposures, microbial interspecies competition or commensalism, and hygiene behaviors. Knowledge of the composition of the human-associated microbiota is increasing, as molecular techniques identify species that were previously undetectable by culture.3,4
The majority of vaginal bacteria are thought to originate from intestinal microbiota in a natural ascension independent of hygiene. Before puberty, the vaginal microbiota is dominated by anaerobic Gram-negative rods and Peptostreptococcus; includes moderate amounts of viridans streptococci, actinomyces, staphylococci, Fusobacterium spp., coliforms, Enterococcus and coryneforms; and contains low frequencies of lactobacilli, Gardnerella vaginalis, Prevotella bivia, Mycoplasma hominis, and yeast.5
During the menarchal period, estrogen regulates the deposition of glycogen on the vaginal epithelium. Glycogen is metabolized by vaginal epithelial cells to glucose, which serves as a substrate for lactobacilli, promoting their growth. Lactobacilli convert glucose to lactate, which lowers the vaginal pH.6 Correspondingly, the most frequent members of the vaginal microbiota of reproductive-age women belong to the genus Lactobacillus, first isolated by Doderlein in 1894 and described by Beijerinck in 1901.7,8Lactobacillus acidophilus was identified as the vaginal “Doderlein bacillus” by Thomas in 1928 with biochemical methods and this species along with L. fermentum was presumed the dominant species until molecular techniques revised the taxonomy of Lactobacillus.9 Currently, the dominant vaginal lactobacilli detected vary with the molecular techniques used, which comprise gene probes, restriction enzyme analysis, randomly amplified polymerase chain reaction, fingerprinting, and pulsed-field electrophoresis. Some techniques require a culturing step, while others allow for direct detection of nonculturable bacteria (Table 1).10–15 Across the various techniques and populations, L. crispatus, L. iners, L. jensenii, and L. gasseri seem to be prevalent.
The shift to a lactobacillus-dominant microbiota in menarchal women is also accompanied by an increased percentage of G. vaginalis, M. hominis, P. bivia and yeasts, all of which decrease postmenopause but can be restored by supplemental estrogen therapy.16–19Escherichia coli, enterococcus, viridans streptococci, and staphylococci remain relatively constant through the life cycle. The vaginal microbiota changes not only with life cycle events but also can fluctuate on a daily basis. Gardnerella and Bacteroides morphotypes on Gram stain can be shown to dominate over lactobacilli transiently for days, especially around the time of menses, or more chronically in the case of bacterial vaginosis (BV). More recently, semiquantitative molecular methods have detected fastidious and unculturable species that also dominate when the normal microbiota is disrupted, particularly Atopobium vaginae.20–24
Bacterial Vaginosis and Sexually Transmitted Infections
BV is a syndrome defined by symptoms and signs of a white, homogenous, malodorous discharge, vaginal itching, increase in vaginal pH above 4.5, development of a fishy odor when 10% KOH reacts with an altered organic acid pattern (including increases in putrescine, cadaverine, and trimethylamine), and vaginal epithelial cells observed on wet mount to be stippled with adherent small rods or cocci (“clue cells”). Three of 4 (discharge, pH, odor, clue cells) clinical or laboratory signs are required for BV diagnosis by the Amsel criteria.25 The relative changes in bacterial concentrations have been tracked by Nugent scoring, which uses staining and microscopy to grade the predominance of 3 morphotypes: lactobacilli, small gram-variable rods or Gram-negative rods (G. vaginalis, Bacteroides), and curved gram-variable rods.26 Currently, molecular techniques are providing new ways to categorize the change in composition of the vaginal microbiota.27 The predominance of G. vaginalis, Bacteroides spp., and A. vaginae in BV is also accompanied by increases in other anaerobes, such as prevotella, mobiluncus, genital mycoplasmas, and an unfolding community of unculturable bacteria but not necessarily a decrease in the geometric mean concentration of lactobacilli.28,29
Nugent score analysis, culture and molecular methods reveal that not all women lacking lactobacillus by culture have BV and most BV cases are asymptomatic.
However, a lack of hydrogen peroxide-producing lactobacilli is associated with BV10,30–34 and may explain why the incidence of recurrent BV is common after antibiotic treatment.35–37 Indeed, risk factors for BV and loss of colonization by hydrogen peroxide-producing lactobacilli include antibiotic usage, douching, frequent sexual intercourse, exposure to a new sex partner, and number of male partners.32,38–42 It is uncertain whether the transmission of a partner factor such as a bacterial species or a bacteriophage that perturbs the lactobacillus population is involved in the etiology of BV.43–46
The exact causes of BV are not known, but certain populations seem to be more at risk. In the United States, BV is more common in black women than in white women47–49 and BV prevalence can be up to 50% in some African populations.50–53 Black women are less likely to be colonized with lactobacilli than white women, and this difference cannot be explained by health behaviors or vaginal pH.54,55 Whether it can be explained by differences in environmental exposure or mother-to-infant transmission of strains is unknown. Infant acquisition of maternal lactobacilli can be demonstrated but appears transient.56,57 In trials, women lacking hydrogen peroxide-producing vaginal strains were colonized spontaneously11; however, it is unknown if the source of these strains was environmental or from cryptic intestinal sources.
Although BV is associated with sexual intercourse and a partner factor may be one cause of BV,58 BV is not classically defined as an STI. However, increased HIV shedding37,52,59–64 trichomonas,65 gonorrhea,66 chlamydia,37,67,68 and herpes simplex virus (HSV)-269,70 infections have been associated with BV. Prevention of BV may therefore reduce the incidence of more than 1 STI. Unfortunately, there is a high incidence of recurrent BV despite standard-of-care treatment with antibiotics.71 The association of hydrogen peroxide-producing lactobacilli with lower BV risk has attracted the interest of therapeutically colonizing women with probiotic lactobacilli to prevent BV.
Materials and Methods
Our primary search strategy sought articles that included treatment or prevention of BV, STIs, or HIV with probiotics. The primary search was conducted on Pubmed and MEDLINE (Ovid) dating from 1960 to December 2006. Our search combined the following terms with and without medical subject headings: BV, treatment, prevention, probiotic, probiotics, sexually transmitted diseases, STIs, Lactobacillus, lactobacilli, and HIV. Review of article references and personal communications with authors from publications produced further references. This search strategy identified 1397 titles of articles. From the 1397 articles, we identified 16 that included clinical trials of probiotics for treatment or prevention of BV or STIs and excluded review articles, editorials, articles focused on gastrointestinal diseases and other uses of probiotics, and articles focused on the treatment of candidal vaginitis. The remaining articles were considered in preparing the background material for the review, including preclinical studies. Articles were grouped according to the major focus of the study or article: selection, safety, and clinical trials. Clinical trials were further divided into dairy-based products, oral pure-culture treatments, vaginal pure-culture treatments, and acceptability studies.
Selection of Probiotic Bacteria for Promotion of Vaginal Health and Prevention of Diseases
The exploration of new urogenital “probiotic” approaches to prevent BV has been prompted by a number of factors including the high incidence of BV in women of African descent, the increased risk BV places these women for other STIs, particularly HIV, the high recurrence of BV with current antibiotic treatments and associated side effects including subsequent yeast vaginitis superinfection, the association of hydrogen peroxide-producing lactobacilli with lower BV risk, and the possibility of genetically modifying probiotic bacteria to inhibit specific STIs. Although the exact causes of BV are unknown, selection of species and strains of therapeutic bacteria have been based on inherent or engineered properties that are likely to maintain a lactobacilli-dominant microbiota and inhibit pathogens. Probiotic strains under current investigation have been chosen based on several characteristics that might accomplish this goal including pathogen binding or exclusion, lactic acid production, hydrogen peroxide production, production of other bacterial metabolites, and modification of host immunity.
Generally, probiotic bacteria should be of human origin, safe, able to transit the digestive system if necessary and able to colonize the target surface.72 Persistence of probiotic bacteria at the vaginal mucosa would be an advantage to reducing recurrent BV or for long-term protection from STIs but has been elusive in most studies. Probiotic bacteria may competitively compete for pathogen-binding sites on mucosal surfaces or produce compounds that prevent pathogen binding.73 Orally administered probiotics may be able to compete with potential pathogens in the gastrointestinal tract and prevent ascending infections, but it is believed that most potential urogenital bacteria need strong vaginal binding properties as well.74 Testing of lactobacillus strains in vitro for suitability as probiotics shows a wide range of adhesiveness of the lactobacilli to the exfoliated vaginal epithelial cells75,76,77 Several collagen-binding proteins have been isolated by surface-enhanced laser desorption/ionization studies, including a 29-kd protein that not only binds lactobacilli to collagen on vaginal epithelial cells but also inhibits significant numbers of pathogens from binding to surfaces.78,79 Both glycoproteins and carbohydrates may be involved in vaginal epithelial cell binding and pathogen exclusion.80,81
Lactobacilli and other probiotic bacteria are also being genetically modified to produce specific HIV inhibitory proteins, both membrane-bound and secreted. A strain of L. jensenii was engineered to produced functional CD4, the primary receptor for HIV.82 There are several classes of proteins that bind to the mannose residues of HIV, including a unique 11 kd protein from cyanobacteria (Nostoc ellipsosporum) called cyanovirin-N and mannose-binding lectins which also bind to Neisseria gonorrhoeae.83 The human commensal bacterium Streptococcus gordonii, L. lactis, L. plantarum, and L. jensenii have been genetically engineered to express functional, HIV-binding cyanovirin-N.84–86 An E. coli strain which colonizes the colon and rectum, and may thus potentially prevent anal transmission, has been modified to secrete peptides hybridized with hemolysin A, a protein that can complex with the HIV fusion protein gp41.87 FI-1, FI-2, and FI-3 are also peptides that can interfere with gp41 and were cloned into L. plantarum and L. gasseri.88L. reuteri RC-14, which has been studied as a potential probiotic, was modified to produce 3 HIV entry and fusion inhibitors: CD4D1D2IgKLC, MIP-1β, and T-1249.89 Antibody to the cellular adhesion molecule intercellular adhesion molecule has been shown to inhibit cell-mediated transepithelial HIV-1 transmission in vitro and was functionally excreted by a bioengineered strain of L. casei.90 Some of these products have been used in rodent models, but none has been tested in humans.
By their production of lactic acid, lactobacilli may help maintain a low vaginal pH that can inhibit other bacteria and viruses. Among postmenopausal women receiving estrogen replacement therapy, those who were lactobacilli-positive had low pH (4.4 ± 0.4) compared with the lactobacilli-negative women (5.2 ± 0.3, P = 0.02).19 In 55 menarchal women, colonization with β-hemolytic streptococci, G. vaginalis, or mixed organisms was associated with higher vaginal pH than colonization with normal microbiota and yeast.91 As previously mentioned, BV is associated with a lack of hydrogen peroxide-producing lactobacilli. Colonization with hydrogen peroxide producers is associated with lower frequency of gonorrhea.92 Compared with women colonized with hydrogen peroxide-producing lactobacilli, women colonized with hydrogen peroxide nonproducers or without lactobacilli had unadjusted odds ratios of HSV-2 seroconversion of 2.4 and 2.6, respectively.70 The difference in seroconversion rates may be due to a direct antiviral effect of hydrogen peroxide or due to an increased risk of HSV infection in women with BV.
Several mammalian peroxidases, including myeloperoxidase, eosinophil peroxidase, and lactoperoxidase can combine with hydrogen peroxide and a halide (chloride, iodide, bromide, thiocyanate) to form “a powerful antimicrobial system” effective against many pathogens.93 Lactobacilli, streptococci, and pneumococci can release hydrogen peroxide and in vitro, in mixed cultures, their toxicity against other bacteria, fungi, viruses, spermatozoa, and tumor cells can be boosted by addition of a peroxidase and a halide. This system was found to inhibit cell-free HIV replication in culture in the presence of hydrogen peroxide-producing, but not nonproducing, lactobacilli.93,94L. crispatus and L. jensenii inhibit gonococci at both acidic and neutral pH. This inhibition is susceptible to bovine catalase, suggesting that hydrogen peroxide is the primary mediator.95 Lactobacilli have been found to produce many other bacteriocins, enzymes, and antimicrobial peptides that may make them more competitive in the vaginal microbiota.96,97,98,99,100,101
Another potentially beneficial characteristic of probiotic bacteria studied in the gastrointestinal tract is their pleomorphic effect on host mucosal immunity that could affect the vaginal mucosa’s defense against HIV and other STIs or resistance to BV.102,103 Evidence for enhancement of humoral responses to rotavirus and Salmonella typhi has been shown through IgA levels in probiotic treated children and adults.104–107 Animal and human data suggest that probiotic use is associated with induction of innate and cell-mediated immune responses,75,108–110 including increased macrophage phagocytic activity,111–113 complement and reticuloendothelial activation,114,115 stimulation of interferon-γ, interleukin (IL)-12, and IL-18,116–119 and increased natural killer cell activity.120 However, the major cell wall component of lactobacilli, muramyldipeptide, can be pyrogenic, and there is concern that lactobacillus enhancement of the T-helper-1 proinflammatory pathway could have negative health consequences.121,122 Also, there is concern that the dosage and duration of therapy must be considered so as to optimally enhance and not suppress immunity.123 Fortunately, probiotic strains have not been found to cause a systemic antibody response.124
Modification of intestinal mucosal immunity by orally administered probiotics may also affect vaginal mucosal immunity to specific pathogens.125 In fact, genetic engineering of probiotic bacteria to express pathogen antigens and serve as oral vaccines is being explored.126–128 In other mucosal vaccine trials, introduction of antigens from HPV and HIV has been accomplished in S. gordonii (an oral commensal) and L. casei, and local and systemic immune responses were detected in BALB/c mice and Cynomolgus monkeys after vaginal colonization with these strains.129 However, there is not yet a good understanding of the relationship between mucosal immunity and BV. In a study of adolescents, BV was found to be inversely associated with lactobacilli counts but was independent of lactobacilli-specific immune responses in isolated peripheral blood leucocytes, and independent of local immune responses measured by antibodies and cytokines measured in cervicovaginal lavages.34 In a study of pregnant women, carriage of a variant of the toll-like receptor-4 gene compared with carriage of the dominant genotype was associated with higher vaginal pH and a 10-fold increase in vaginal G. vaginalis levels. Colonization with G. vaginalis or anaerobic Gram-negative rods in the dominant allele carriers was associated with elevated vaginal IL-1 and IL-1ra but not in the variant homozygotes.130 Probiotic modulation of mucosal immunity may help prevent BV and other STIs, but it is difficult to predict which strains might do this without knowing the immune correlates of protection and how specific strains will affect these factors.
The use of viable organisms for therapy requires special safety considerations because, unlike conventional therapeutics, the dose given can increase with bacterial growth. The bacteria introduced may become part of and compete with the normal microbiota, and normal microbiota can be opportunistically pathogenic, particularly in immunocompromised individuals. Conversely, most probiotic organisms are selected from the normal microbiota with which the host is already adapted. In the case of urogenital probiotics, the route of application may be oral or vaginal, and each route has separate safety issues and may require different dosages. The majority of safety data on probiotics comes from gastrointestinal uses introduced orally.
Lactobacilli are normally present in the fluids of the oral cavity (103–104 cfu/g), the ileum (103–107 cfu/g), the colon (104–108 cfu/g), and the vagina (107–108 cfu/g).131,132 Natural cases of invasive infection due to lactobacilli and bifidobacteria are extremely rare and are estimated to represent 0.05% to 0.4% of cases of infective endocarditis and bacteremia.133L. rhamnosus and L. paracasei subsp. paracasei are the species most associated with endocarditis, but many species have been found to produce glycosidases and proteases that might enable them to colonize the heart.134 In addition, as mentioned previously, the major cell wall component of lactobacilli, muramyldipeptide, can be pyrogenic.121,122 The dosage and duration of therapy must be considered so as to optimally enhance and not suppress immunity.123 Overall, however, adverse effects have not been found in a large healthy population receiving Lactobacillus GG in Finland.135,136
Most cases of invasive disease and bacteremia by lactobacilli have been described in patients with significant morbidities such as cancer, diabetes, postoperatively, and after liver transplantation,.135,137,138 In most cases, the lactobacilli were not those associated with probiotic strains; however, a case report describes a liver abscess due to a L. rhamnosus strain indistinguishable from L. rhamnosus strain GG.139,140 Two clinical studies have been conducted to assess the safety of oral probiotics in small groups of specific immunocompromised patients (e.g., patients with HIV infection), and the findings of these studies support the safety of probiotics consumed by such groups.141,142 Preterm infants who received lactobacillus GG did note have side effects, and S. boulardii given orally has been well tolerated in different populations, including patients with AIDS and Crohn’s disease.143–145 Another case report describes L. casei pneumonia and sepsis in a patient with AIDS.146
A special concern exists regarding the use of enterococci as probiotics because of possible acquisition of multiple antibiotic resistance by pathogenic bacteria, including resistance to vancomycin. This concern could also apply to genetically modified lactobacilli or other bacteria if the plasmid encoding their transgene also contains an antibiotic resistance gene for laboratory selection.144,147–149 Transgene products must also be tested for immunogenicity to rule out the possible development of allergic reactions, induction of tolerance to pathogens, or induction of autoimmunity.
Clinical Trials of Probiotics for Treatment or Prevention of Recurrent BV
Probiotics are already in popular use for urogenital applications, mostly with dairy products or food supplements, but many of these products lack appropriate species designations, do not contain a list of species, contain extra species, or vary in concentrations of microorganisms.150,151 One study examining dairy products found a high degree of contamination with unwanted species including Enterococcus faecium, Clostridium sporogenes, Streptococcus mitis, and Pseudomonas species. All products in the study contained viable lactobacilli, 62% of which produced hydrogen peroxide but had a questionable ability to colonize the vagina.152 A comparison of in vitro adherence to vaginal epithelial cells showed that lactobacilli isolated from yogurt had significantly lower adherence than clinical isolates or American Type Culture Collection cultures.153
Studies investigating the ability of dairy-based lactobacilli to colonize the vagina include a randomized, controlled study of 24 healthy Japanese women at 35 weeks of gestation who were orally given 120 g/d of fermented milk containing 109 colony forming units/mL of L. johnsonii La1 (Nestle Japan, Tokyo, Japan) or placebo-fermented milk for 2 weeks. Administration of the probiotic food significantly increased the number of vaginal lactobacilli (P = 0.025).154 In contrast, a study of L. rhamnosus GG, a hydrogen peroxide producer which lacks the 29 kd biosurfactant protein, showed that when given orally once or twice a day to 42 healthy postmenopausal women for 1 month, only 9.5% of the women had even low-level vaginal colonization, although 78.6% demonstrated gastrointestinal colonization.155
Trials testing dairy products for treatment of BV include 2 noncontrolled trials from a Japanese group, 1 using lactobacillus of unknown designation in yogurt among 11 women, and the other among 16 women using Enterococcus faecalis T-110, Clostridium butyricum TO-A, and Bacillus mesentericus TO-A (“Bio-three”). In both studies, a 3-day intravaginal treatment with the test product was followed by lower vaginal pH, decreased symptoms, and “bacteriological eradication” in about half of the subjects.156,157 In an open randomized trial, 61 women clinically diagnosed with BV were treated vaginally for 7 days with 1 of 4 treatments: acetic-acid-impregnated jelly, “Dionoestrol” cream, a commercially available yogurt based on L. acidophilus of unknown designation, or metronidazole. Only the metronidazole treatment showed significant resolution of BV 4 weeks later (in 13/14 patients), whereas the yogurt group showed resolution in only 1/14 patients.158 A randomized control trial compared daily ingestion of yogurt with live L. acidophilus with ingestion of a pasteurized yogurt control in a cross-over design among 46 women with recurrent BV, candida vaginitis, or both. Only 7 of the 42 patients completed the study, but interim treatment time points at 1 and 2 months showed significantly increased positive vaginal cultures for L. acidophilus (P <0.05) and significantly decreased episodes of BV in the live L. acidophilus treatment group (P <0.001). No difference was seen in candidal vaginitis or candidal colonization, but the candida results may have been confounded since 20% of patients were treated with topical antifungals.159 In another study, yogurt containing L. acidophilus, of unknown designation, was delivered by douche to 32 pregnant women and compared with the use of an acetic acid impregnated tampon in a 32-member control group. There were significantly fewer women in the yogurt-treated group with BV at 1 month and 2 months posttreatment compared with the acetic acid tampon-treated group (P <0.05) or a third, untreated control group (P <0.0005).160
Oral Pure-Culture Treatments.
One of the more extensively studied probiotic lactobacillus strains, L. rhamnosus GR-1 (formerly L. casei), is a urogenital tract isolate that was chosen after comparison with 34 other strains for size, adherence, and exclusion of uropathogens to uroepithelial cells, and inhibition of uropathogen growth.161
L. rhamnosus has been studied in combination with another strain, L. fermentum RC-14, which was found to have strong adhesion to uroepithelial cells, possibly mediated by an 29 kd “biosurfactant” protein that also inhibits pathogen binding.79,162,163L. fermentum RC-14 also produces significant amounts of hydrogen peroxide, whereas L. rhamnosus GR-1 does not. These strains have been studied for survival of gastrointestinal transit without significant increases in IgG, IgA, IgM, IL-2, IL-4, IL-6, interferon-γ,164 and can be recovered from the vagina after oral administration.165–169
In 1 study, 42 healthy women were randomized to 3 different daily oral dosing regimens of L. rhamnosus GR-1 plus L. fermentum RC-14, or a single daily oral dose of L. rhamnosus GG (a probiotic with evidence for gastrointestinal use) for 28 days. A greater percentage of women taking a dose of 1.6 × 109 L. rhamnosus GR-1/L. fermentum RC-14 in a twice daily regimen had a normal Nugent score at 2 weeks posttreatment than women taking 1 × 1010L. rhamnosus GG once a day (P = 0.017), but no statistical difference was seen with the highest dose of 6 × 109L. rhamnosus GR-1/L. fermentum RC-14 taken once daily.170 A randomized, placebo-controlled trial of 64 healthy women given daily oral capsules of L. rhamnosus GR-1 and L. fermentum RC-14 for 60 days showed significant increase in Lactobacillus counts at days 28 and 60 of treatment, significant decrease in yeast counts at day 28, and significant decrease in coliform counts at days 28, 60, and 90 (30 days posttreatment) in the lactobacilli-treated versus placebo control groups (P <0.05).171 In a separate randomized, placebo-controlled trial of 59 healthy women given daily oral capsules of L. rhamnosus GR-1 and L. fermentum RC-14 or placebo for 60 days, a significantly improved outcome in the treatment when compared with the control arm (P = 0.008).172
Most recently, a trial in Nigeria randomized 125 women with BV to twice-daily oral capsules of L. rhamnosus GR-1 and L. fermentum RC-14 or placebo for 30 days with all participants receiving metronidazole 500 mg twice daily for the first 7 days of the experimental treatment. A significantly greater number of women in the treatment group compared with the placebo group had a normal vaginal microbiota, a negative sialidase test, and absence of discharge or odor at day 30 (P <0.001). However, this trial had a much greater loss to follow-up in the treatment group compared with the controls (25% vs. 5%), which may have biased the results.173
Vaginal Pure-Culture Treatments.
One early descriptive study tested a vaginal douche of an undesignated strain of L. acidophilus isolated from a healthy woman by colony morphology, Gram stain and biochemical tests. A douche of 108 to 109 bacteria diluted from broth into hypertonic saline was applied vaginally twice daily for 1 or 2 weeks by 38 women aged 15 to 57 who presented with complaints of pain, pruritus, secretions, odor, or inflammation and who failed previous topical or systemic antibiotics. The patients also took an unspecified oral B-vitamin complex daily under the rationale that lactobacilli require B vitamins for growth. At 1 week posttreatment, 76% of the women had what was considered “normal microbiota,” and 55% had normal microbiota at 36-month follow-up compared with 13% at baseline.174
A product called Vivag, a hydrogen peroxide-producing strain of L. acidophilus, was tested in 60 Swedish women with BV in a double-blind placebo-controlled trial. Twice daily use of a vaginal suppository for 6 days led to a 57% improvement in BV compared with no improvement in the pure-starch placebo group at 7 to 10 days after the start of treatment (P <0.005), with no difference between groups after the next menstrual period or 20 to 40 days after start of treatment.175 Another product containing a hydrogen peroxide-producing strain of L. acidophilus plus 0.03 mg estriol called Gynoflor was tested in 32 nonmenopausal women who had intermediate microbiota or BV by Nugent criteria in a randomized placebo-controlled trial. At 2 and 4 weeks after a 6-day treatment with 1 to 2 vaginal tablets daily, the lactobacilli-treated group had significantly fewer cases of BV and significantly greater lactobacilli colonization compared with the placebo group (P <0.05 for all outcomes and time points).176
L. crispatus CTV-05 is a vaginally derived hydrogen peroxide-producing strain being studied as a potential urogenital probiotic, which can colonize the vagina and be detected among other species by molecular techniques.11 In a placebo-controlled, randomized control trial, the results of which have been available on the Internet or in meeting proceedings, women with BV were randomized to L. crispatus CTV-05 or placebo intravaginal capsule twice daily for 3 days monthly for 3 months. There was no significant improvement in the cure rate for BV when CTV-05 was administered with a single oral 2 g dose of metronidazole in all subjects. However, the subset of subjects who received CTV-05 and were colonized with L. crispatus CTV-05 had a significantly higher clinical cure rate at 30 days than those who were not colonized. Furthermore, this phase 2/3 study of L. crispatus CTV-05 demonstrated the product to be safe.177–179
In a randomized control trial of 255 Swedish women with clinical or microbiologic criteria for BV, all subjects were treated with vaginal clindamycin ovules for 3 days before randomization to using tampons impregnated with a mixture of pure cultures of L. gasseri, L. casei var rhamnosus, and L. fermentum, or placebo tampons during the following menstrual period. Roughly, equal numbers of women were excluded in the 2 arms, leaving 187 women for analysis. There was no statistical difference between the 2 groups in the intention-to-treat analysis or the included sample when comparing rates of clinical cure, improvement, partial improvement or failure. Bacteriological cure rates were also not significantly different. The study did not include L. crispatus in the treatment and used only 106 colony forming units per tampon.180
A nonblinded study of 40 Nigerian women selected from a cohort of 350 women with BV used a undefined and unvalidated randomization method to assign women to 2 groups based on a priori knowledge of age, history of previous urogenital infections, and severity of current BV infection. One group received 2 gelatin capsules containing L. rhamnosus GR-1 and L. reuteri RC-14 at bedtime vaginally for 5 days. The other group applied 0.75% metronidazole gel vaginally twice a day for 5 days. There was a significantly greater cure rate in the probiotic group at day 6 (P = 0.016) and day 15 (P = 0.002) and a trend toward significance at day 30 (P = 0.056)181 (Table 2).
As for microbicides and other female-controlled STI interventions, it is important to know whether probiotics would be an acceptable product for women, particularly in developing countries. A randomized control trial of L. crispatus vaginal capsule or placebo included an analysis of satisfaction among 232 of the participants. The study found satisfaction and positive beliefs about use of the probiotic capsule were prevalent, especially among women with a clinical and microbiologic response to BV therapy, but even those without response were willing to use the product again.179 In a study of receptivity for probiotic products among 280 premenopausal female students in an African university in Nigeria, 82% of the subjects stated that they would use probiotics orally or as vaginal capsules to improve vaginal health. Over one-third (36%) of women indicated they would be willing to use the probiotics as part of their daily self-care.182 Another study examined a millet-based fermented beverage as a possible culturally appropriate vehicle for probiotic administration in Ghana.183 Several commercial probiotic products for gastrointestinal use have already been introduced in South Africa, but studies have shown poor correlation between the labeled organisms and actual contents.184
The high rate of BV women in general and in African populations in particular, the association between BV and risk of contraction of STIs, the association between BV and loss of hydrogen peroxide-producing lactobacilli, and the increasing acceptance of lactobacilli probiotics for gastrointestinal uses make urogenital use of lactobacilli attractive female-initiated strategy for treatment of BV, and possibly prevention of STIs. The fundamental difficulty with attempting this approach is the lack of knowledge of what the root cause of BV is, and thus whether supplementation with lactobacilli will help. Many confounding issues also remain, such as the exact interrelationship between BV, HIV, and HSV-2. The current incidence of HIV infection in developing countries creates an urgent need for testing novel female-controlled approaches in clinical trials, particularly the ones that have previous evidence of safety.
Previous studies suggest that well characterized lactobacilli that produce substantial amounts of hydrogen peroxide and adhere well to vaginal epithelial cells may be able to colonize the vagina and have the best chance of treating BV and potentially preventing female STI and HIV acquisition. The existing studies on this topic are intriguing but do not yet determine whether probiotic lactobacilli administered vaginally or orally would be better than current antibiotic treatment alone to prevent long-term BV recurrence. A sufficiently powered, well-designed, randomized placebo-controlled trial of a product will be required to measure the efficacy of a probiotic to augment antibiotic treatment to prevent recurrent BV. If results from such a trial demonstrate efficacy, future studies should be designed to determine whether a probiotic could significantly lower the risk for STIs and HIV infection in at-risk female populations. Although still at the experimental stage, development and testing of engineered probiotics that could deliver locally specific molecules to inhibit HIV and other pathogens, should be pursued with careful ethical considerations, as it could provide a simple, cheap and sustainable prevention tool to women in the future. Although significant hurdles in this field remain, ongoing development and testing of probiotics should become a greater priority as a means to mitigate the growing global STI and HIV epidemics.
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