Application of Lactobacillus iners in assessing the prognosis of bacterial vaginosis

Abstract

The present invention discloses the application of Lactobacillus iners in evaluating the prognosis of bacterial vaginosis. The present invention provides the application of the relative abundance of Lactobacillus iners in the vagina as a marker in the preparation of a product for evaluating the prognosis of bacterial vaginosis. The present invention has found that the prognosis of bacterial vaginosis patients with a higher relative abundance of Lactobacillus iners in the vagina before treatment is significantly better than that of bacterial vaginosis patients with a lower relative abundance of Lactobacillus iners in the vagina before treatment after conventional metronidazole treatment. The present invention is of great significance for further refinement of diagnosis and treatment of bacterial vaginosis.

Description

Technical Field

[0001] The invention relates to the field of biomedicine, and in particular to application of Lactobacillus iners in evaluating the prognosis of bacterial vaginosis. Background Art

[0002] Bacterial vaginosis (BV) is the most common lower genital tract infection in women of childbearing age. Changes in the composition of the normal vaginal microecological flora, namely a decrease in the dominant lactobacillus that maintains the normal acidic environment of the vagina, and an increase in other microorganisms (mainly anaerobic bacteria) are the main causes of BV, but its specific cause is still unclear. Bacterial vaginosis can increase women's susceptibility to pelvic inflammatory disease, premature birth, HPV infection, and a variety of sexually transmitted diseases. Currently, metronidazole and clindamycin are used clinically to treat BV according to standard regimens. Previous studies have reported that the cure rate of these two drugs is 48% to 96%, but recent studies have shown that the treatment failure rate of the two drugs has increased significantly.

[0003] There is no report on predicting the prognosis of BV treatment based on the composition of vaginal lactobacilli before treatment. Summary of the invention

[0004] The purpose of the invention is to provide an application of Lactobacillus iners in evaluating the prognosis of bacterial vaginosis.

[0005] In a first aspect, the present invention claims the use of the relative abundance of Lactobacillus iners in the vagina as a marker in the preparation of a product for assessing the prognosis of bacterial vaginosis.

[0006] In a second aspect, the present invention claims the use of a substance for detecting Lactobacillus iners in the preparation of a product for assessing the prognosis of bacterial vaginosis.

[0007] Among them, the substance is capable of detecting the relative abundance of Lactobacillus iners in the vagina.

[0008] Furthermore, the substance may be a primer pair or an antibody capable of specifically detecting Lactobacillus iners.

[0009] If necessary, the substance may also be primers, reagents and / or instruments required for 16S rDNA sequencing of bacterial genomes.

[0010] According to needs, the substance may also contain a standard substance; the standard substance may be, for example, Lactobacillus iners or its 16SrDNA sequence.

[0011] In the above two aspects, the evaluation of the prognosis of bacterial vaginosis is to evaluate the treatment effect of metronidazole on patients with bacterial vaginosis.

[0012] In a specific embodiment of the present invention, the patient with bacterial vaginosis is treated for 5 consecutive days by vaginal administration of 5% metronidazole gel (ie, 5g of metronidazole per 100g of finished gel).

[0013] In a third aspect, the present invention claims a system for assessing the prognosis of bacterial vaginosis.

[0014] The system for assessing the prognosis of bacterial vaginosis claimed in the present invention may include:

[0015] (A1) Reagents and / or instruments;

[0016] The reagent and / or instrument has the following functions: detecting the relative abundance of Lactobacillus iners in the vagina;

[0017] (A2) device;

[0018] The device includes a data input module, a threshold storage module, a data comparison module, and a judgment module;

[0019] The data input module is configured to input (A1) the relative abundance value of Lactobacillus iners in the vagina of the patient with bacterial vaginosis to be tested before treatment;

[0020] The threshold storage module is configured to store a threshold; the threshold is 0.2975%.

[0021] The data comparison module is configured to receive the value obtained by the detection (A1) sent by the data input module, call the threshold value in the threshold value storage module, and compare the relative abundance value of Lactobacillus iners in the vagina of the patient with bacterial vaginosis before treatment with the threshold value;

[0022] The judgment module is configured to receive the comparison result sent by the data comparison module, and then make a result judgment as follows: if the relative abundance value of Lactobacillus iners in the vagina of the patient with bacterial vaginosis to be tested before treatment is greater than the threshold, then the patient with bacterial vaginosis to be tested has or is a candidate for having a good prognosis; if the relative abundance value of Lactobacillus iners in the vagina of the patient with bacterial vaginosis to be tested before treatment is less than or equal to the threshold, then the patient with bacterial vaginosis to be tested has or is a candidate for having a poor prognosis.

[0023] Among them, the prognosis of the bacterial vaginosis patient to be tested whose relative abundance value of Lactobacillus iners in the vagina before treatment is greater than the threshold is significantly better than the prognosis of the bacterial vaginosis patient to be tested whose relative abundance value of Lactobacillus iners in the vagina before treatment is less than or equal to the threshold.

[0024] In the system, the prognosis assessment of bacterial vaginosis is to assess the treatment effect of metronidazole on patients with bacterial vaginosis.

[0025] In a specific embodiment of the present invention, the patient with bacterial vaginosis is treated for 5 consecutive days by vaginal administration of 5% metronidazole gel (ie, 5g of metronidazole per 100g of finished gel).

[0026] In a fourth aspect, the present invention claims the use of metronidazole in the preparation of a medicament for treating patients with specific bacterial vaginosis.

[0027] The specific bacterial vaginosis patient is a bacterial vaginosis patient whose relative abundance value of Lactobacillus iners in the vagina before treatment is greater than 0.2975%.

[0028] In a fifth aspect, the present invention claims protection for a kit of products for screening and treating patients with specific bacterial vaginosis (ie, a diagnostic and treatment kit).

[0029] The complete set of products for screening and treating patients with specific bacterial vaginosis (i.e., complete set of diagnostic and therapeutic products) claimed in the present invention is composed of the following:

[0030] (B1) a substance capable of detecting the relative abundance of Lactobacillus iners in the vagina (for screening);

[0031] (B2) Metronidazole (for treatment);

[0032] The specific bacterial vaginosis patient is a bacterial vaginosis patient whose relative abundance value of Lactobacillus iners in the vagina before treatment is greater than 0.2975%.

[0033] In the present invention, the relative abundance refers to the ratio of Lactobacillus iners to the total number of bacteria in the vagina.

[0034] In a specific embodiment of the present invention, the term "before treatment" specifically refers to before treatment with metronidazole.

[0035] The present invention has found that the prognosis of patients with bacterial vaginosis who have a relatively high relative abundance of Lactobacillus iners in the vagina before treatment is significantly better than that of patients with bacterial vaginosis who have a relatively low relative abundance of Lactobacillus iners in the vagina before treatment after conventional metronidazole treatment. The present invention is of great significance for further refinement of diagnosis and treatment of bacterial vaginosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is the phylogenetic tree of the four Lactobacillus genomes involved in the experiment.

[0037] Figure 2The results of the relative abundance test of Lactobacillus crispatus in the vagina of different subjects before and after treatment. The asterisks in the figure indicate that the relative abundance of specific bacteria in the two groups is significantly different. * indicates 0.01<p≤0.05; ** indicates 0.001≤p<0.01; *** indicates 0.0001≤p<0.001; **** indicates p<0.0001.

[0038] Figure 3 This is the ROC curve obtained by the present invention. DETAILED DESCRIPTION

[0039] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0040] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0041] Example 1: Application of Lactobacillus iners in assessing the prognosis of bacterial vaginosis

[0042] 1. Cohort recruitment and sample collection

[0043] Samples were collected from August 2020 to August 2021, and 308 HIV-negative, non-pregnant women of childbearing age (18-50 years old) who visited Peking University First Hospital for vulvovaginal discomfort and / or abnormal vaginal discharge, and 62 women who went to Peking University First Hospital for gynecological examinations were the research subjects. All patients were tested for HIV, HPV, HSV-2, syphilis, Chlamydia trachomatis, Neisseria gonorrhoeae, vulvovaginal candidiasis, Trichomonas vaginalis, Ureaplasma urealyticum, Mycoplasma hominis, and urinary tract infection. Patients infected with any of the above pathogens or diagnosed with any medical disease (such as hypertension, diabetes, hyperlipidemia, etc.) were excluded from this study. Other exclusion criteria include: multiple sexual partners, history of intrauterine operation (such as intrauterine contraceptive device insertion, hysteroscopy, etc.), menstruation at the time of sampling, use of any type of antibiotics in any way within 30 days before sampling, and sexual intercourse within 7 days before sampling. At the same time, we recorded the participants' menstrual cycle, last menstrual period, and reproductive history. After screening, 138 patients who met the criteria were included in the follow-up study. At the same time, 41 healthy women (18-50 years old) without any disease were included in the negative control group.

[0044] The studies involving human participants were reviewed and approved by the Ethics Committee of Peking University First Hospital. All participants gave written informed consent for the publication of any potentially identifiable images or data included in this article and agreed to voluntarily participate in our follow-up.

[0045] 2. Sample Collection and Diagnostic Procedures

[0046] During the gynecological examination, two vaginal secretion specimens were collected from the upper 1 / 3 of the anterior vaginal wall of each participant using a disposable sterile vaginal swab (brand: Becton, Dickinson and Company), one of which was used for DNA extraction and subsequent sequencing procedures, and the other was used for Gram staining for vaginal secretion microscopy. The former was properly stored at -80°C immediately after collection until use, while the latter was Gram stained immediately after collection and independently inspected by two experienced testing technicians to ensure the accuracy of the experiment.

[0047] The specific diagnostic criteria for BV are as follows:

[0048] 1) The diagnosis is based on the Nugent score of the patient's vaginal secretions stained with Gram stain: 0-3 points for BV negative; 4-6 points for BV intermediate; 7-10 points for BV positive.

[0049] 2) Diagnosis is based on the "Amsel criteria" proposed by Amsel et al. in 1991, that is, BV diagnosis requires at least three of the following four criteria to be met:

[0050] a) Vaginal discharge is large in volume and thin in texture;

[0051] b) vaginal pH>4.5;

[0052] c) Amine test positive (a fishy odor can be smelled after adding 10% potassium hydroxide aqueous solution (i.e., 10g of potassium hydroxide per 100g of potassium hydroxide aqueous solution) to vaginal secretions);

[0053] d) Clue cells account for >20% in saline wet mount.

[0054] Only when the patient meets the diagnostic criteria of 1) and 2) at the same time, the patient is diagnosed with BV.

[0055] III. Treatment and Follow-up

[0056] All patients diagnosed with BV (a total of 129 cases) were given 5% metronidazole gel (Nimexin, 5g metronidazole per 100g flat gel) vaginally for topical treatment for 5 days and were required to return to the obstetrics and gynecology clinic for a follow-up examination within 1 week after the end of treatment and repeat the test steps in step 2. The clinical treatment outcome criteria for patients were as follows:

[0057] 1) Patients with a reexamination Nugent score of 0-3 were judged as “cured” (Group C);

[0058] 2) Patients with a reexamination Nugent score of 4-6 were judged as “intermediate BV” (group I);

[0059] 3) Patients with a reexamination Nugent score of 7-10 were judged as “uncured” (Group F).

[0060] IV. Sample DNA Extraction and Sequencing

[0061] The vaginal swab samples that were well stored at -80°C were restored to room temperature and centrifuged at 1000g for 10 minutes. ProKit (Qiagen) kit was used to extract DNA from the sample according to the steps provided in the kit.

[0062] The V3 forward primer 5′-CCTACGGGNBGCASCAG-3′ and the V4 reverse primer 5′-GACTACNVGGGTATCTAATCC-3′ (N in the primer sequence represents A or T or C or G; B represents G or T or C; S represents G or C; V represents G or A or C) were used to amplify the highly variable V3 and V4 segments in the 16SrDNA gene fragments in the samples, and were checked and analyzed on 2% agarose gel. 250bp double-end sequencing data were obtained using the Illumina NovaSeq6000 platform. The original offline data were spliced ​​using Full Length Adjustment of SHort Reads (FLASH) (Magoc and Salzberg, 2011) software, with the minimum Overlap set to 100bp and the maximum Overlap set to 250bp. The fastq_quality_filter command of FASTX-Toolkit (for specific methods, please refer to: http: / / hannonlab.cshl.edu / fastx_toolkit / ) was used to perform quality control on the spliced ​​sequences and filter out low-quality sequences. After quality control, the sequences were compared with the GOLD database (created by EgdarRC et al. in a 2011 study) and chimeras were removed using the UCHIME command of the USEARCH software. The minimum number of sequences of all samples was used as the standard (6555 sequences) to level the data of each sequencing sample. The sequences after leveling were annotated with species using the Ribosomal Database Project (RDP) Classifier software to determine the classification level of the sequences, namely (Kingdom), Phylum, Class, Order, Family, and Genus.

[0063] In addition, we obtained the 16S rDNA sequences of four lactobacilli, Lactobacillus crispatus, Lactobacillus iners, Lactobacillus jensenii, and Lactobacillus gasseri, from the NCBI database. The phylogenetic tree confirmed that the 16S rDNA sequence could distinguish the four lactobacilli ( Figure 1 ) A database was constructed using the 16S rDNA sequences of the four lactobacilli, and the above-leveled sample sequences (6555 sequences) were aligned using BLASTn, with identity>=99% as the cutoff, to calculate the relative abundance of the four lactobacilli in different samples.

[0064] The relative abundance refers to the number of reads containing Lactobacillus iners (L. iners) detected in the sample by the BLAST program divided by the total number of reads (6555), which reflects the ratio of Lactobacillus iners (L. iners) to the total number of bacteria in the subject's vagina.

[0065] 5. Statistics

[0066] The “ggpubr” package in the R language program (version number: v4.1.1) was used to perform Wilcoxon test, Kruskal-Wallis test and Mann-Whitney test within and between different groups to compare the differences in the abundance of different Lactobacilli between groups, and SPSS software (version number: 26) was used to draw the ROC curve.

[0067] 6. Results and Analysis

[0068] The results showed that the patients in the cured group had a higher relative abundance of Lactobacillus iners than those in the other two groups before treatment (7.31% vs 2.51%, p = 0.0021; 7.31% vs 2.86%, p = 0.0274). However, the three bacteria Lactobacillus crispatus, Lactobacillus jensenii and Lactobacillus gasseri did not show the above pattern. For detailed results, please refer to Tables 1, 2 and Figure 2 .

[0069] Table 1. Results of determination of Lactobacillus iners abundance in the vagina of each subject before treatment and comparison of effects after treatment

[0070]

[0071]

[0072]

[0073]

[0074]

[0075]

[0076] Note: Subject numbers marked with C indicate the cured group after metronidazole treatment; those marked with I indicate the intermediate BV group after metronidazole treatment; those marked with F indicate the uncured group after metronidazole treatment; and those marked with H indicate healthy people.

[0077] Table 2. Data analysis of the results shown in Table 1

[0078]

[0079] Based on the above results, the ROC curve was drawn in a binary classification manner according to the C group (i.e., the cured group after metronidazole treatment) and the non-C group (i.e., the I group that turned to the intermediate type after metronidazole treatment and the F group that was not cured), and the point with the best sensitivity and specificity was found from the ROC curve. The relative abundance value of Lactobacillus iners corresponding to the point with the best sensitivity and specificity was used as the threshold. After testing, BV patients whose relative abundance value of Lactobacillus iners in the vagina was greater than the threshold were judged to have a good prognosis after metronidazole treatment, and BV patients whose relative abundance value was less than or equal to the threshold were judged to have a poor prognosis after metronidazole treatment. The final ROC curve can be found in Figure 3 , and its corresponding AUC value is 0.841. The corresponding threshold is 0.2975%, that is, BV patients with a relative abundance value of Lactobacillus iners greater than 0.2975% in the vagina are judged to have a good prognosis after metronidazole treatment, and BV patients with a relative abundance value less than or equal to 0.2975% in the vagina are judged to have a poor prognosis after metronidazole treatment.

[0080] The experimental results of the present invention show that metronidazole has a better therapeutic effect on patients with a relative abundance value of Lactobacillus iners in the vagina greater than 0.2975%. The present invention has a strong guiding role in the selection of clinical treatment drugs for BV.

[0081] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. A system for assessing the prognosis of bacterial vaginosis, comprising: (A1) Reagents and / or instruments; The reagent and / or instrument has the following functions: detecting the relative abundance of Lactobacillus iners in the vagina; (A2) Device; The device includes a data input module, a threshold storage module, a data comparison module, and a judgment module; The data input module is configured to input the relative abundance value of Lactobacillus iners in the vagina of the patient with bacterial vaginosis to be tested before treatment obtained by (A1) detection; The threshold storage module is configured to store a threshold; the threshold is 0.2975%; The data comparison module is configured to receive the value obtained by the detection (A1) sent by the data input module, call the threshold value in the threshold value storage module, and compare the relative abundance value of Lactobacillus iners in the vagina of the patient with bacterial vaginosis before treatment with the threshold value; The judgment module is configured to receive the comparison result sent by the data comparison module, and then make a result judgment as follows: if the relative abundance value of Lactobacillus iners in the vagina of the patient with bacterial vaginosis to be tested before treatment is greater than the threshold value, the patient with bacterial vaginosis to be tested is or is a candidate for having a good prognosis; if the relative abundance value of Lactobacillus iners in the vagina of the patient with bacterial vaginosis to be tested before treatment is less than or equal to the threshold value, the patient with bacterial vaginosis to be tested is or is a candidate for having a poor prognosis; The prognosis assessment of bacterial vaginosis is to assess the treatment effect of metronidazole on patients with bacterial vaginosis.

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