Treatment strategies in different phenotypic forms of bacterial vaginosis

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BACKGROUND

Bacterial vaginosis (BV) is the most prevalent disease, affecting 23%–29% of women of reproductive age [1]. The traditional definition is dysbiosis, i.e., a disruption of the normal balance of the vaginal microbiota, with a massive increase of facultative and obligate anaerobic bacteria (mainly Gardnerella spp.) and a loss of lactobacilli. This definition accurately describes the change in the microbiota; however, it does not explain the underlying pathophysiology, leaving unanswered the question of how to treat BV, including refractory and recurrent BV.

Unfortunately, in addition to local dysbiosis of the vaginal microflora, BV is associated with various medical conditions. For example, BV patients are at a 1.53-fold higher risk for pelvic inflammatory disease (PID) and a 3.32-fold higher risk for infertility. In pregnancy, BV increases the risk for preterm birth by a factor of 2.16 and for late miscarriage by a factor of 6.32 as a result of ascending infection [2–4]. Furthermore, BV promotes co-infection with sexually transmitted infections (STIs) such as Chlamydia trachomatis, Mycoplasma genitalium, Neisseria gonorrhoeae, Trichomonas vaginalis, human papillomavirus, and human immunodeficiency virus [5].

Until recently, Gardnerella vaginalis (G. vaginalis) was thought to be the only species in the Gardnerella genus. However, genetic differences within the genus have now been identified, resulting in 13 species of Gardnerella spp, including G. vaginalis, G. piotii, G. leopoldii, and G. swidsinskii, along with nine additional unnamed species [6]. According to recent research, several species of Gardnerella spp. co-occur in BV biofilm [7].

In approximately half of the cases, patients do not report symptoms despite changes in the vaginal microbiota that are characteristic of BV. The disease is characterized by symptoms such as increased homogeneous vaginal discharge that produces an unpleasant fishy odor, as well as vulvar irritation, dyspareunia, and dysuria. Signs of inflammation (redness, swelling, and pain) are usually absent [4].

In the past 20 years, polymerase chain reaction (PCR) tests and sequencing techniques, as well as fluorescence in situ hybridization (FISH), have been evaluated for the diagnosis of BV. In contrast to the reference methods, these techniques are able to map changes to the vaginal microbiota both quantitatively and on the taxonomic level [8]. In Russia, the most informative diagnostic modality in the outpatient practice of an obstetrician-gynecologist is PCR with hybridization and fluorescence detection, which allows for estimation of the ratio of the total number of bacteria and lactobacilli, as well as opportunistic microorganisms associated with BV (Gardnerella vaginalis, Atopobium vaginae) in the vaginal biotope [9].

Anaerobic microorganisms play a crucial role in the development of BV. Consequently, clinical protocols recommend 5-nitroimidazole or lincosamide agents such as metronidazole or clindamycin as first-line therapy [10, 11]. Treatment-refractory or recurrent BV involving at least three episodes per year causes impaired quality of life in >65% of affected individuals [12]. The high rate of treatment failure is attributable to several factors. First, the therapeutic effect of antibiotics on polymicrobial biofilms dominated by certain Gardnerella species is insufficient. Second, these biofilms are resistant to metronidazole. Third, the biofilms are unable to recolonize the vagina with lactobacilli, which results in re-infection from sexual partners [13]. Consequently, alternative therapeutic agents effective against biofilm (antiseptics, natural antimicrobials, plant extracts, lactic acid-glycogen complex, suppositories containing live Lactobacillus acidophilus) are being evaluated as monotherapy or as an adjunct to antibiotic therapy for BV [4, 14–16].

Given the incidence of BV, the variety of its complications in gynecological and obstetrical practice, and the high rate of treatment failure, there is an urgent need to develop new laboratory and therapeutic approaches, as well as methods of reliable prevention of the disease.

The aim of the study was to identify species of vaginal Lactobacillus spp. and Gardnerella vaginalis genotypes in women prior to treatment of BV and following therapy with a one-step antibacterial regimen versus a two-step therapy with suppositories containing at least 10⁷ live Lactobacillus acidophilus or a lactic acid-glycogen complex.

MATERIALS AND METHODS

The following inclusion criteria were used to define the study groups: female patients aged 18 to 45 years, including those diagnosed with BV according to clinical criteria (presence of vaginal discharge, elevated pH of vaginal contents above 4.5) and positive aminotest confirmed by PCR.

After signing a voluntary informed consent to participate in the study, women were divided into three groups based on subsequent treatment. Group 1 patients received antibiotic therapy alone (n = 30), Group 2 patients were recommended antibiotic therapy and suppositories containing at least 10⁷ live Lactobacillus acidophilus (n = 30), and Group 3 patients received antibiotic therapy in combination with lactic acid and glycogen (n = 30).

Exclusion criteria: inability or unwillingness to participate in the study or fulfill the requirements of the study; age below 18 and above 45 years; premature ovarian failure, early or surgical menopause; acute pelvic inflammatory disease; pregnancy or lactation, taking antibacterial agents in the previous three weeks; concomitant STIs (chlamydia, gonococcal infection, trichomoniasis, or infection caused by M. genitalium) and/or other infection requiring additional antibacterial and/or antimycotic therapy; indication of intolerance to the lactic acid-glycogen complex in the history; severe liver function disorders with signs of hepatic insufficiency; diabetes mellitus and severe somatic diseases that may affect the study and interpretation of its results; mental disorders that do not allow interpretation of the study results; and malignant neoplasms of any localization.

At the stage of inclusion in the study, the patients underwent a gynecological examination. The pH of vaginal contents was measured using indicator strips (Kolpo-test pH, Russia). A vaginal fluid sample was collected from the upper third of the lateral vaginal vault for a comprehensive test. This test is a multiplex PCR system that is comparable in diagnostic performance to international criteria for the detection of BV.

In the subsequent visit four weeks after treatment, the patients’ complaints were re-evaluated. In addition, a gynecological examination was performed together with pH measurement of vaginal contents; vaginal fluid from the upper third of the lateral vaginal vault was collected for PCR testing.

Treatment efficacy was evaluated according to the following criteria: the absence of homogeneous whitish-gray vaginal discharge with malodor, pH of the vaginal fluid less than 4.5, and a decrease in the titer of opportunistic microorganisms and normocenosis according to the results of a PCR test.

The statistical processing of the data was performed using Statistica 8.0 software for mathematical and statistical analysis. The nature of the distribution was assessed using the Kolmogorov–Smirnov and Shapiro–Wilk criteria. Spearman’s rank correlation analysis was used to assess the strength and direction of correlation between ordinal and nominative data (significance level was considered reliable at p < 0.05).

RESULTS

Initially, all patients presented with complaints of white (77.7%) and gray (22.2%) vaginal discharge in scarce (12%), moderate (58%), and abundant (30%) amounts, accompanied by itching (27%), burning (14%), dysuria (6%), dyspareunia (12%), and malodor (60%).

The mean age of the women studied was 32.1 ± 3.6 years.

There were no statistically significant differences between the groups in the frequency of somatic and gynecological disorders.

The most frequently used method of contraception among the patients was interrupted sexual intercourse (48%), while 37% and 15% of the patients employed barrier and hormonal methods (mainly combined oral contraceptives), respectively.

Upon examination, white discharge was observed in 55% of patients, gray discharge in 45%, and malodor in 40%, with no evidence of hyperemia or swelling of the vaginal mucosa.

The average pH of vaginal discharge was 6, ranging from 5.5 to 6.5.

At the second stage, patients were divided into three groups of 30 individuals based on their treatment regimens. A specialized laboratory test was then conducted to ascertain the species diversity of G. vaginalis and Lactobacillus spp. within the vaginal biotope. The nucleic acid amplification method was employed to identify the four most prevalent species of vaginal lactobacilli (L. crispatus, L. iners, L. jensenii, and L. gasseri) and five species of G. vaginalis (G. vaginalis genotype 1, G. vaginalis genotype 2, G. swidsinskii, G. leopoldii, and G. vaginalis genotype 3), and the totality of indicators was subsequently evaluated after treatment.

The presence of G. vaginalis DNA was identified in the vaginal biotope of all subjects via PCR-based diagnostics. No statistically significant differences were observed between groups in the number of detections, suggesting that the study groups were comparable in this regard. A thorough examination of species diversity revealed that in Group 1 patients G. vaginalis type 2 (24.32%) and G. vaginalis type 3 (8.1%) were identified less frequently, while a significantly elevated level of G. leopoldii was detected in Group 2 patients (66.6%).

The most prevalent species among Lactobacillus spp. in patients from all groups was Lactobacillus iners, accounting for 60% of cases (see Figure 1).

 

Fig. 1. PCR data of patients with bacterial vaginosis before treatment.

 

Conversely, Lactobacillus crispatus and Lactobacillus jensenii were identified with a lower frequency in Group 3 patients, at 30.76% and 28.3%, respectively.

In the four weeks following treatment, as indicated by the control examination, patients across all groups exhibited predominantly moderate mucous discharge without malodor.

The pH value of vaginal discharge 28 days after treatment ranged from 4.5 in Groups 1 and 3 to 4.0 in Group 2, with an average of 4.25.

No adverse reactions, such as itching and burning in the vagina, were detected in any patient during treatment or by the time of the repeat examination.

A PCR test was conducted to evaluate the species diversity of G. vaginalis and Lactobacillus spp. within the vaginal biotope following treatment.

The complete disappearance of Gardnerella was not observed; however, a numerical decrease in G. vaginalis type 1 and G. vaginalis type 2 with an increase in G. leopoldii and G. vaginalis type 3 in all groups was observed. A decrease in the prevalence of Lactobacillus iners was observed in 59.45% and 53.00% of patients in Groups 1 and 3, respectively. Conversely, Group 2 exhibited an increase in the number of Lactobacillus iners among 62.96% of patients. Many researchers associate an increased number of Lactobacillus iners with recurrent BV. A decline in the number of Lactobacillus jenseni was observed (48.64%, 11.53%, and 37.14% in Group 1, 2, and 3, respectively). However, this decline was more statistically significant in Group 2 (p <0.05). When evaluating Lactobacillus gasseri, a decrease was observed in Group 1 (up to 13.51%) and Group 2 (up to 22.22%), with a slight increase in Group 3 (26.92%). An increase in the number of Lactobacillus crispatus was observed in all groups (up to 56.75%, 85.18%, and 46.15%, respectively), but the increase was more statistically significant in Group 2 (p <0.05). According to many researchers, an increased value of Lactobacillus crispatus is associated with a ”healthy” vaginal microbiome. The results obtained one month after treatment are presented in Figure 2.

 

Fig. 2. PCR data of patients with bacterial vaginosis after treatment.

 

The laboratory results indicated that all treatment groups exhibited clinical efficacy following antibiotic monotherapy (particularly clindamycin) and two-component therapy (an antibiotic complex with lactic acid and glycogen, or suppositories containing at least 10⁷ live Lactobacillus acidophilus). However, Group 2 demonstrated a more substantial reduction in the species of G. vaginalis and L. iners, accompanied by an increase in L. crispatus. The validity of the second stage of BV therapy, as well as the selection of pharmaceutical agents, remains a subject of debate. The controversy surrounding the efficacy of live Lactobacillus acidophilus in recovering healthy vaginal microbiota, as opposed to the complex of lactic acid and glycogen to create an acidic environment conducive to the proliferation of lactobacilli, remains unresolved.

The findings suggest genetic diversity of microorganisms in the vaginal biotope; however, it is unknown whether these results are associated with the use of pre- and probiotics. Further follow-up is necessary to assess long-term outcomes and develop recommendations for the treatment of BV.

CONCLUSION

A comparative analysis was conducted to assess the efficacy and safety of suppositories containing Lactobacillus spp. and a lactic acid-glycogen complex following antibiotic therapy, with the aim of preventing the recurrence of BV. The analysis took into account the species identification of Lactobacillus spp. and Gardnerella vaginalis genotypes isolated from the vaginal samples of the study participants. Further research is necessary to evaluate the long-term outcomes of BV treatment.

ADDITIONAL INFO

Authors’ contributions. Yu.E. Dobrokhotova edited the manuscript; V.D. Kazantseva collected and reviewed study data, processed statistical data, wrote the text; L.A. Ozolinya developed study concept and design, wrote the text; T.N. Savchenko collected and reviewed study data, wrote the text. All authors confirm that their authorship meets the international ICMJE criteria (all authors have made a significant contribution to the development of the concept, research and preparation of the article, read and approved the final version before publication).

Funding source. This study was not supported by any external sources of funding.

Competing interests. The authors declares that there are no obvious and potential conflicts of interest associated with the publication of this article.

Consent for publication. Written consent was obtained from all the study participants before the study screening in according to the study protocol approved by the local ethic committee.

Ethical statement. The study was performed within the framework of the dissertation of Valeria Dmitrievna Kazantseva and was approved by the local ethical committee of N.I. Pirogov Russian National Research Medical University (extract from protocol No. 213 dated 13 December 2021).

About the authors

Yulia E. Dobrokhotova

Pirogov Russian National Research Medical University

Email: pr.dobrohotova@mail.ru
ORCID iD: 0000-0002-7830-2290
SPIN-code: 2925-9948

MD, Dr. Sci. (Medicine), Professor

Russian Federation, Moscow

Valeriya D. Kazantseva

Pirogov Russian National Research Medical University

Author for correspondence.
Email: shapee08@mail.ru
ORCID iD: 0000-0002-4011-3195
SPIN-code: 6973-6276

Graduate Student

Russian Federation, Moscow

Lyudmila A. Ozolinya

Pirogov Russian National Research Medical University

Email: ozolinya@yandex.ru
ORCID iD: 0000-0002-2353-123X
SPIN-code: 9407-9014

MD, Dr. Sci. (Medicine), Professor

Russian Federation, Moscow

Tatyana N. Savchenko

Pirogov Russian National Research Medical University

Email: 12111944t@mail.ru
ORCID iD: 0000-0001-7244-4944
SPIN-code: 3157-3682

MD, Dr. Sci. (Medicine), Professor

Russian Federation, Moscow

References

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