RU2364340C1 - Method of biological material sampling for microbial analysis of vaginal microflora - Google Patents

Abstract

FIELD: medicine; gynaecology.

SUBSTANCE: method of cervicovaginal secretion sampling involves introduction of a device into posterior vaginal vault. Said device comprises a flexible polypropylene tube of overall length 275 mm with a hole of external diametre 2.6 mm on a tip. Along the full length of the tube there is a movable piston. Said device been introduced is used to aspirate the vaginal secretion.

EFFECT: more reliable quantitative assessment of vaginal biotope due to bacteria sampling from parietal and luminal regions in adequate volume, preserved viability of facultatively and obligate anaerobes in micro-oxic environment in the tube that is generated in sampling process, possibility of accurate determination of seed volume.

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Description

The invention relates to medicine, namely to gynecology and obstetrics, and can be used to collect biological material from the vagina in order to conduct bacteriological studies, which allow to determine the specific and quantitative composition of vaginal microflora in women with vaginal dysbiosis.

Reliable laboratory confirmation of dysbiotic changes in the vaginal biocenosis can be obtained as a result of a cultural study of cervico-vaginal secretion (Ankirskaya AS, Bacterial vaginosis. Obstetrics and gynecology. - 2005. - No. 3. - P.10-13).

To date, the dominant role of opportunistic bacteria in the infectious pathology of the vagina has been established: it is shown that the same microorganisms can be the causative agents of “nonspecific” (opportunistic) infections under the same conditions, and under the physiological norm they can be an integral part of indigenous microflora. (Ankirskaya A.S., Muravyova V.V. Vaginal infections: diagnosis. Laboratory diagnosis of opportunistic infections of the vagina. Consilium-medicum, 2005. - Volume 7. - No. 3). In this regard, the level of contamination of the vagina by opportunistic microorganisms is important for the choice of tactics for the management and treatment of patients with opportunistic vaginal infections. The exact quantitative microbiological characteristics of the vaginal microflora are largely determined by the method of taking biological material.

There is a universal method for producing material for gene-diagnostic testing of the vaginal microflora of large groups of women, which consists in taking biological material of the vagina and conducting gene-amplification analysis of cellular DNA from a hygienic tampon in contact with the vagina for at least 6 hours and then placed in an airtight polymer a container with a liquid preservative that stops the growth of bacteria, and during gene-amplification analysis, the swab is squeezed into a container Here 4-5 times to complete the flushing of cells (patent RU 2209040, 2003).

Known methods for collecting material potentially containing vaginal microflora consist in taking the vaginal discharge with the mucosa with a sterile cotton swab probe, microbiological loop (V. Menshikov. Clinical and laboratory analytics. Private analytical technologies in the clinical laboratory. M .: Agat-Med, 2003. - Volume 4. - P.310-314), cervical cytobrush (Voropaeva EA, Afanasyev SS, Kudryavtseva MV and others. Microecology and indicators of humoral immunity in women with nonspecific genital diseases. Journal mick obiol, 2005. -. №3 -. S.65-69).

The prototype of the invention is a method of collecting cervico-vaginal secretions using a bacteriological (calibration or microbiological) loop (Menshikov VV Clinical and laboratory analytics. Private analytical technologies in a clinical laboratory. M .: Agat-Med, 2003. - Volume 4. - S.310-314).

When sampling the vaginal discharge from the posterior fornix using a sterile cotton swab probe, cervical cytogen brush, it is likely that an insufficient volume of material is obtained, which can lead to false negative results in the diagnosis of vaginal dysbiosis. The amount of biological material in this case is to some extent determined by its consistency in case of vaginal dysbiosis, which can vary: with a homogeneous watery discharge, the hygroscopic properties of a cotton swab, cytobrush can hold a greater amount of secretion than in the case of thick or “curdled” secretions. Moreover, the reduced exposure time of the swab probe, cervical cytobrush in the posterior vaginal fornix also contributes to the receipt of the material not in full. The results of bacteriological studies of the material obtained by these methods give an exclusively qualitative or semi-quantitative assessment of the state of vaginal microflora.

When sampling material using bacteriological loops, each of which has a standard diameter that allows you to transfer a certain amount of fluid to the meniscus (fluid volume with a relatively constant number of microorganisms), an insufficiently accurate quantitative microbiological characteristic of the vaginal microecosystem is obtained due to the fact that in some cases the density and viscosity of the vaginal discharge excludes the possibility of the formation of a meniscus and, therefore, the standard dosage of material a.

The disadvantage of the above methods of taking the vaginal discharge is the lack of the possibility in the process of obtaining material to create conditions with a low oxygen content necessary for the survival of facultative and obligate-anaerobic microorganisms that prevail during dysbiotic shifts in the vaginal microsystem (Kira E.F. Bacterial vaginosis. St. Petersburg. : OOO "Neva-Lux", 2001. - S.114-123).

The objective of the invention is to develop an effective method of taking biological material from the vagina for bacteriological research.

EFFECT: increased reliability of bacteriological studies of vaginal microflora by taking bacteria of the parietal and luminal regions in sufficient quantities, preserving the viability of facultative and obligate-anaerobic microorganisms under conditions of low oxygen content in the tube created in the process of taking the material, and the ability to accurately determine the seed dose .

The proposed method is illustrated by the following drawings: figure 1 shows a General view of the used device for the fence in assembled form; figure 2 - fence vaginal discharge using the device.

The proposed method is as follows. The collection of biological material from the vagina is carried out before manual examination after the introduction of mirrors from the posterior fornix or pathologically altered sections of the mucous membrane. A device is used that contains a flexible transparent polypropylene tube 1 with a total length of 275 mm, on the working end 2 of which there is a hole 3 with an outer diameter of 2.6 mm, and a plastic piston 4 is movably placed inside the tube along the entire length, which is designed to create negative pressure during material intake In addition, it is used to push the extracted material into a test tube. Without touching the walls of the vagina, the device is inserted into the posterior arch and, holding it with one hand, the piston is moved by pulling with the other, as a result of which a vacuum is formed, and the released cavity of the tube is filled with biological material. Subsequently, the device is rotated 2-3 times around the longitudinal axis in the lateral directions in order to collect the maximum volume of material. Moving the internal piston to the working end, empty the tube and place the resulting material in a test tube with a transport medium or buffer solution.

The proposed method for producing biological material from the vagina provides a sufficient number of bacteria of the parietal and luminal region. Negative pressure in the tube creates favorable conditions (with a reduced oxygen content in the medium) for the survival of facultative and obligate-anaerobic microorganisms in the process of taking the material. In addition, the method of taking material using this device allows you to get the exact volume of the vaginal discharge to determine the initial ratios between associates in microbial pathobiocenoses, thereby increasing the reliability of the bacteriological method in the diagnosis of opportunistic infections of the vagina.

We give an example of clinical use of the proposed method.

Patient A., born in 1980, turned to an obstetrician-gynecologist with complaints of abundant pus from the genital tract for two days, a burning sensation and itching in the vagina after unprotected sexual intercourse.

From gynecological diseases: cervical ectopy - cryodestruction at 23 years. Of the common diseases: chronic gastroduodenitis, stage of remission. Menstrual function without features. There was no pregnancy history. Sexual life since 19 years, out of wedlock. During the last 4 years of marriage, a married couple has been using a condom for contraception.

On examination, the general condition is satisfactory.

Status genitalis: External genitalia are developed correctly, female type hair. Inspection data in the mirrors: the cervix of the uterus. There is hyperemia and swelling of the mucous membrane of the vagina and cervix, vaginal discharge in an increased amount, yellowish, homogeneous consistency, viscous, odorless. Per vaginum: The vagina is narrow. The vaults are free, symmetrical. The body of the uterus in anteversio-flexio, normal in size, in the midline, is dense, mobile, painless. Appendages on both sides are not palpable, their area is painless.

In order to establish a diagnosis, the following diagnostic measures were carried out: a smear was taken from the posterior vaginal fornix, epithelial scraping from the cervical canal and urethra for PCR diagnostics, a detachable from the posterior fornix of the vagina was obtained by the proposed device and microbiological loop (3 mm in diameter) for bacteriological examination.

In a Gram smear: white blood cells in large quantities in the SC, moderate epithelial cells, gram-variable flora in large numbers, “key” cells were not found. M.hominis, U.urealyticum, C.trachomatis, T.vaginalis, N..gonorrhoeae from the cervical canal, urethra, were not detected by PCR. As a result of the examination, a preliminary diagnosis was established: Acute nonspecific vaginitis.

The bacteriological data obtained by taking the material with the proposed device and microbiological loop differed significantly (table 1): in the first case, higher levels of seeding of the vaginal biocenosis by opportunistic microorganisms were revealed and the true picture of the severity of the dysbiotic process in the vagina was reflected.

The patient was given antibacterial treatment according to the results of a bacteriological study.

The proposed method for producing biological material from the vagina was used in 73 patients, which significantly increased the reliability of bacteriological studies of vaginal microflora and introduced it more widely into clinical practice.

Table 1. Comparative quantitative and specific characteristics of the vaginal microflora of patient A, depending on the method of obtaining biological material for bacteriological research Type of microorganisms Seeding rate (lg CFU / ml) vaginal discharge fence of the proposed method vaginal discharge fence with a calibrated loop (3 mm in diameter) Lactobacillus spp. 9 7.84 S. agalactiae 11,4 6.6 E. faecium 7.17 4.92 A. viridans 6.35 not displayed G. vaginalis 3 not displayed

Claims (1)

A method of collecting a cervico-vaginal secretion, comprising introducing a device into the posterior vaginal fornix, characterized in that a device is introduced containing a flexible polypropylene tube with a total length of 275 mm, at the working end of which there is an opening with an external diameter of 2.6 mm, and inside the tube throughout the piston is movably placed along the length, and the vaginal discharge is aspirated.

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