*Điểm số Nugent là một phương pháp đánh giá vi khuẩn Gardnerella vaginalis trong tài liệu vô khuẩn học của phụ nữ. Phương pháp này được sử dụng để đánh giá mức độ nhiễm trùng âm đạo và giúp xác định liệu có cần điều trị bệnh hay không.
Phương pháp này dựa trên việc đánh giá số lượng các loại tế bào khác nhau trong một mẫu bệnh phẩm, bao gồm tế bào biểu mô âm đạo, tế bào phân bào cấu trúc và tế bào thượng bì âm đạo. Từ đó, một điểm số được tính toán để đánh giá mức độ nhiễm trùng âm đạo. Điểm số Nugent càng cao thì mức độ nhiễm trùng càng cao và ngược lại.
Thông thường, nếu điểm số Nugent là 7 đến 10 điểm, thì có thể cho rằng mức độ nhiễm trùng âm đạo là cao và cần điều trị bệnh. Tuy nhiên, quyết định liệu có cần điều trị hay không cũng phải xem xét kết hợp với các dấu hiệu và triệu chứng của bệnh như mùi hôi, ngứa và rát âm đạo. Vì vậy, việc quyết định điều trị bệnh nên được thực hiện bởi bác sĩ chuyên khoa phụ khoa sau khi đã đánh giá toàn diện các tình trạng và triệu chứng của bệnh nhân.
Effectiveness of the Two Microorganisms Lactobacillus fermentum LF15 and Lactobacillus plantarum LP01, Formulated in Slow-release Vaginal Tablets, in Women Affected by Bacterial Vaginosis
MATERIALS AND METHODS
In Vitro Study
Bacterial Strains and Growth Conditions
The lactobacilli strains used in this study, all isolated either from the feces of healthy humans or from vaginal swabs of healthy female subjects, were classified based on their phenotypic and genotypic characteristics. The G. vaginalis strain employed was purchased from the ATCC.
The in vitro inhibitory activity of different lactobacilli against G. vaginalis was quantified at Biolab Research laboratories (Novara, Italy). Before being used in the experiment, the G. vaginalis ATCC 10231 strain was grown in brain-heart infusion broth (BHI) (Oxoid, Milan, Italy) containing 2% (wt/wt) gelatin, 0.5% yeast extract, 0.1% starch, and 0.1% glucose,30 whereas individual lactobacilli were cultured in liquid de Man, Rogosa and Sharpe (MRS) medium (BD Difco, Milan, Italy).
All the plates were incubated at 37°C for 48 hours under anaerobic conditions (GasPak system) with Anaerocult A kits (Merck Millipore, Milan, Italy).
Assessment of the Antagonistic Activity of Selected Lactobacilli Toward G. vaginalis
Culture supernatants of the different lactobacilli tested (2 L. crispatus, 2 L. paracasei, and 4 L. fermentum) were prepared as follows: an overnight culture of each isolate was centrifuged at 5000g. The resulting supernatant was neutralized at pH 6.5 with NaOH 1 N, sterilized by filtration through syringe filters (Ministart pore size: 0.22 μm), and assayed for the presence of any inhibitory molecule in the broth.
Neutralized supernatants of individual Lactobacillus were then added in different percentages to fresh BHI broth, prepared as described above, inoculated with G. vaginalis. The growth of Gardnerella alone (positive control) and in the presence of different concentrations of neutralized supernatants ranging from 0.5% to 4%, after 24 and 48 hours of incubation at 37°C in microaerophilic conditions, was quantified by means of optical density at 600 nm (OD600). The growth of positive control (Gardnerella alone) was assessed in BHI broth added to a quantity of fresh MRS broth ranging from 0.5% to 4%. Thus, the positive controls were as similar as possible to test tubes inoculated with individual lactobacilli. This test was repeated 3 times for each strain to ensure reliability and reproducibility.
Human Trial
Study Design
Thirty-five female patients (aged between 18 and 50, mean 34.7±8.9, no menopausal women) were initially enrolled in this pilot, randomized, placebo-controlled study between September and October 2012 at the Gynecological Associated Medical Practice “Vicariotto-De Maria” (Milan, Italy). Informed written consent was obtained from all participants involved in the study. This study was carried out in accordance with the Helsinki Declaration (2000) of the World Medical Association.
Eligible subjects were at least 18 years old and had active, symptomatic acute BV according to the Amsel criteria and diagnosed according to the Nugent score,10 involving the microscopic quantitation of bacterial morphotypes yielding a score between 0 and 10.
Patients were excluded from the study if the Nugent score was <7, considered as the threshold of a significant imbalance in the vaginal microbiota composition. Other exclusion criteria were pregnancy, mixed infections, known seropositivity for the human immunodeficiency virus (HIV), and treatment with antibiotic agents or other products containing lactobacilli or bifidobacteria even if taken orally, in the previous 2 months.
After enrollment, subjects were randomly divided into 2 groups, one being given the active formulation (Group A, 24 subjects) and the other a placebo (Group B, 11 subjects). The 2 lactobacilli L. fermentum LF15 (DSM 26955) and L. plantarum LP01 (LMG P-21021) were tested in the form of slow-release vaginal tablets, also containing 50 mg of tara gum. The amount of each strain was 400 million live cells per dose. In addition, each active tablet contained 340 mg of arabinogalactan and 241 mg of fructooligosaccharides (FOS). The active formulation and the placebo were indistinguishable in appearance. In the placebo tablets, the amounts of the 2 lactobacilli, the tara gum, and the 2 fibers arabinogalactan and FOS were replaced by an equivalent quantity of microcrystalline cellulose and anhydrous calcium hydrogen phosphate, 2 inert ingredients.
Each female subject was instructed to apply a vaginal tablet once a day for 7 consecutive nights, preferably before going to bed. Then, to maintain vaginal colonization by the 2 lactobacilli, patients used 1 tablet every 3 days for the following 3 weeks. The acute treatment lasted, therefore, 4 weeks in total. In the following month, to maintain long-term vaginal colonization against possible recurrences, patients used 1 tablet per week.
Patients were prohibited from using topical vaginal or other systemic antibiotic agents at any time during the study. They were also prohibited from using other products containing lactobacilli or bifidobacteria, even if taken orally, throughout the duration of the study.
A clinical examination was performed at the Gynecological Associated Medical Practice “Vicariotto-De Maria” and the Nugent score was quantified for each patient at enrollment (d0), after 28 days (d28), and at the end of the second month of relapse prevention (d56).
Statistical Analysis
Paired and unpaired t test statistical analysis were used to weigh the results and compare them between d28 and d0, d56 and d0, d56 and d28 in each group (paired) as well as between the active group and the placebo at the same time (unpaired). The Nugent score was classified as BV (≥7), intermediate situation (4 to 6), and healthy vaginal microbiota (≤3). Differences were considered significant at P≤0.05.
RESULTS
In Vitro Study
Antagonistic Activity of Selected Lactobacilli Towards G. vaginalis
The in vitro inhibitory results of different lactobacilli towards G. vaginalis ATCC 10231 are shown in Table 1.
L. fermentum LF15 showed the strongest activity after both 24 and 48 hours (82.2% and 88.5% with 4% of neutralized supernatant, respectively). As also previously highlighted by our laboratory, the fresh MRS added to BHI broth boosted the growth of G. vaginalis according to a dose-response, with the highest difference highlighted after 48 hours (0.874 OD600 with 0.5% of MRS and 1.837 OD600 with 4% of MRS).
A dose-dependent growth inhibition was recorded with only some neutralized supernatants, with particular reference to all L. fermentum bacteria after 48 hours. It is also interesting to note that the 2 L. paracasei showed peculiar behavior, with the percentage of inhibition decreasing in parallel with the increase in the neutralized supernatants added to BHI broth. When using 4% of supernatant, the ability to counteract Gardnerella growth was minimal after 48 hours (5.6% and 4.8% with L. paracasei LPC08 and L. paracasei ID 1608, respectively). The results registered with the 2 L. crispatus were relatively similar to the inhibition percentages recorded with the 4 L. fermentum after 48 hours, whereas, after 1 day, L. crispatus ID 1626 showed a weaker inhibition when using 4% of supernatant, probably because the growth promotion induced by MRS offset the antagonistic action of the inhibitory molecules present in the supernatant of the strain.
Human Trial
Efficacy of 2 Lactobacilli in Improving the Nugent Score in Women Diagnosed With BV
Thirty-four female subjects of 35 initially enrolled in the study completed the treatment according to the instructions provided. Only 1 dropout in the placebo group was recorded during the second month, due to protocol deviation and not to adverse events.
An association of the 2 lactobacilli L. fermentum LF15 and L. plantarum LP01 significantly reduced the Nugent score below the threshold of 7 after 28 days in 22 patients of 24 in the active group (91.7%, P<0.001) (Table 2). Eight women (33.3%) recorded a Nugent score between 4 and 6, evidence of an intermediate situation, whereas the remaining 14 (58.3%) showed a score ≤3, an indicator of the restoration of a physiological vaginal microbiota. At the end of the second month, only 4 women registered a Nugent score >7 and definable as BV (16.7%, P=0.065 compared with d28). In the placebo group, no significant differences were recorded at any time. The statistical comparison between the 2 groups (unpaired t test) at the same times highlighted statistically significant differences (P<0.001 at both d28 and d56), whereas at baseline, the subjects were comparable (P=0.137).
Figure 1 shows the average Nugent score recorded in the placebo group and in the group being given the 2 lactobacilli at the different times. At baseline, all the subjects recorded a score >7, with a mean equal to 8.54 in group A and 7.90 in the placebo group (P=0.137). After 28 and 56 days, the mean Nugent score in the placebo group was still >7, whereas in the active group, mean values of 3.50 and 4.25 were recorded, respectively.
DISCUSSION
It is a recognized fact that microbial communities have a robust influence on human health and quality of life. It is not astounding that the bacterial community within the human vagina has a profound impact on a woman’s health, as the microbiota present in this organ plays a crucial role in determining the biochemical profile of the vagina and its inflammatory profile.31
The healthy vagina is populated predominantly by lactobacilli. The species differ with regard to their ability to maintain a stable population through environmental modifications such as pH changes due to sexual intercourse or menstruation and with respect to their ability to counteract the growth of other bacteria.
All of the species produce lactic acid and selected biotypes also secrete hydrogen peroxide and bacteriocins, molecules that are known for their ability to hinder the growth and persistence of specific target bacterial species.
BV is the most common vaginal syndrome afflicting fertile, premenopausal, and pregnant women.31 Furthermore, BV is often asymptomatic, as about 50% of women with this condition have no symptoms at all and the prevalence in apparently healthy women is around 10%. In cases of BV, beneficial lactic acid-producing bacteria, with particular reference to lactobacilli, are replaced by amine-producing anaerobic bacteria. Microbiological analysis of BV has shown the gram-variable bacterium G. vaginalis to be the most frequent causative organism. This bacterium has been detected in >98% of BV cases.
However, it is not clear if some events induce a drop in the population of lactobacilli, which in turn makes local conditions tolerant to the growth of other bacteria, if BV-associated species such as G. vaginalis displace the lactobacilli according to certain kinetics, or whether these 2 factors are not directly related.32
Treatment of BV involves oral or local administration of metronidazole or clindamycin, and varies in efficacy (48% to 85% for absence of infection, ≥4 wk after treatment).33 The overall long-term cure rate is low, as BV recurs in up to 40% of women within 3 months after initiation of antibiotic therapy and in up to 50% of women after 6 months.19 There are several unpleasant side effects and disadvantages associated with these therapies, including superinfections by pathogenic microorganisms18 and susceptibility of lactobacilli to clindamycin,34 a fact that could expose the vaginal milieu to possible reinfections with harmful microbes. Moreover, vaginal pathogens, particularly G. vaginalis and anaerobic bacteria, are showing increased drug resistance.35,36
Many studies have provided evidence of the valuable functions of a healthy microbiota and suggested the selection of bacterial strains, recognized as probiotics, with beneficial effects for the prevention or treatment of conditions involving a disruption of indigenous microbiota.
Lactobacilli use in BV is supported by positive results obtained in some clinical trials. The majority of the studies yielding positive results have been performed using probiotic preparations containing high doses of lactobacilli, thus suggesting that the amount of exogenously applied lactobacilli could have a role in the effectiveness of the product.31
However, it is well known that only selected strains could exert significant effectiveness, not limited to the nonspecific mechanism of action attributable to the secretion of organic acids such as lactic and acetic acids.
This study suggests that L. fermentum LF15 (DSM 26955) and L. plantarum LP01 (LMG P-21021) are able to effectively counteract acute Gardnerella infections and significantly improve the uncomfortable symptoms reported by women with BV. This evidence may be at least partially attributed to the presence of tara gum, an ingredient that is able to create a mechanical barrier against the adhesion of Gardnerella and of other possible harmful microbes to the surface of vaginal mucosa. The overall compliance and tolerability were very good, with only 1 dropout in the placebo group recorded, due to protocol deviation and not to any adverse events.
The very limited percentage of recurrences recorded in the active group during the second month of the protocol suggests a long-term physiological protection thanks to the integration of the 2 lactobacilli into the vaginal microbiota and their adhesion to the epithelial cells of the mucosa. In this regard, the use of arabinogalactan and FOS, 2 fibers that are able to enhance vaginal colonization by the 2 lactobacilli, could have a role in the long-term barrier against reinfections recorded in the second month of the study protocol.
Even though the placebo group included a smaller number of subjects compared with the active group, the placebo effect was very low, as no significant statistical differences were recorded either after 28 days (P=0.619) or at the end of the protocol (P=0.823), thus suggesting that the improvement recorded in the subjects being given the 2 lactobacilli is specific and attributable to the overall effect of the product.
It is also interesting to point out the fact that an in vitro functionality exerted by a selected Lactobacillus, such as the inhibition of Gardnerella growth by means of specific mechanisms of action, was successfully confirmed during this pilot trial in female subjects diagnosed with BV. This could suggest that the in vitro antagonism toward G. vaginalis could be valuable as a criterion to effectively select lactobacilli with a protective action in cases of BV, both in terms of an acute treatment and for long-term prevention of recurrences. In the in vitro part of the study, the selection of a proper growth medium suitable for the inhibition test for Gardnerella was another key point, as presumably some components present in MRS, the medium commonly used to grow lactobacilli before the inhibition assessment, could potentially promote the growth of Gardnerella. In fact, the growth of Gardnerella in BHI alone was relatively poor, especially after 48 hours.
In the light of the in vitro inhibitory activity against E. coli exerted mainly by L. plantarum LP01,29 the prospective use of its association with L. fermentum LF15 in aerobic vaginitis (AV) could prove interesting. In fact, AV is predominantly associated with E. coli, but S. agalactiae and S. aureus are also involved. Standard treatment of AV consists of oral or intravaginal antibiotics, although these cannot automatically restore normal flora characterized by a high concentration of lactobacilli, thus facilitating relapses and reinfections.
In conclusion, this study investigates a possible innovative approach aimed at counteracting acute vaginal microbiota imbalance and preventing possible recurrences either caused by G. vaginalis (BV) or by E. coli (aerobic vaginitis), although future additional investigations will be needed to quantify the effectiveness of L. fermentum LF15 and L. plantarum LP01 in that condition.
Nguồn: https://journals.lww.com/jcge/Fulltext/2014/11001/Effectiveness_of_the_Two_Microorganisms.28.aspx
