Diagnostic technique for papilloma virus infection

FIELD: medicine.

SUBSTANCE: there is used for diagnostics of transient and persistent latent papilloma virus infection. The diagnostic technique for papilloma virus infection is ensured by history taking and integrated clinical-laboratory examination. As anamnestic signs, there are assumed compromised oncologic characteristics inheritance, early sexual life, age younger than 20 years old and promiscuity; as clinical - anogenital warts, erosion and ectopic neck of uterus, contact hemorrhagic diathesis, Ovuli nabotti, inflammatory diseases of small pelvis organs and intrauterine spiral. Herewith the laboratory signs are sexually transmitted diseases, mixtinfection, deficient lactic acid bacilli, vaginal disbiosis caused by conditional-pathogenic flora, bacterial vaginosis, virus-virus associations, urogenital herpes, urogenital mycoplasma infection, urogenital ureaplasma infection, urogenital candidiasis, urogenital Chlamidia infection and infection with various genotypes of human papilloma virus. Each sign is scored, and depending the number of points, persistent or transient clinical course of papilloma virus infections, or follow-up examination is performed.

EFFECT: determining tactics of the following management of the patient, forming group of potential risk for development of focal dysontogenetic and malignant transformations of urogenital epithelium.

1 dwg, 2 tbl, 2 ex

 

The invention relates to medicine, diagnostics perspective in the field of venereal diseases and can be used for the diagnosis and prevention of transient and persistent latent papillomavirus infection.

High prevalence of genital human papillomavirus infection, reaching epidemic diseases, its proven role in the development of benign and malignant neoplasms of genital organs, the heterogeneity of the human papillomavirus and polyorgano called pathology indicative not only of biomedical relevance of this problem, but its social value. Currently officially registered only cases of anogenital (venereal) warts [ICD X, And 63.0], intensive the incidence of which in the Russian Federation in 2004-2005 was 32.9-32,1 cases per 100 thousand population.

In the last decade has seen widespread growth of anogenital human papillomavirus (HPV), one of the most common viral sexually transmitted infections. In the world annually more than 30 million cases of anogenital warts. These figures reflect only the frequency of clinical manifestations of human papillomavirus infection, and not the true extent of infection in the population, so as not logged subclinical and latent infection. A huge number of young women with ambiguous clinical picture vaginal condylomata are the reservoir of infection and pose a risk to sexual partners and unborn child. Considering the cost of diagnosis and treatment of all types of human papillomavirus infection (the value of an investment at $ 1.6- $ 6 billion. per year), in the U.S. it is considered the most expensive after infection AIDS (1).

Data on the frequency of human papillomavirus infection of the urogenital tract in Russia incomplete and based on the statistics of individual medical institutions or doctors involved in this pathology (2, 3).

Diagnosis of human papillomavirus infection presents certain difficulties, especially in its latent form, in which despite the presence of human papilloma virus morphological changes in the tissue is not observed (4, 5). The urgency of the problem increases due to the fact that you have the etiologic role of human papillomavirus high carcinogenic risk in the development of cervical cancer

(6-13).

To date there is no clear certainty factors contributing to different rates of progression of papilloma viral infection, there were no significant prognostic signs, contributing to long-term persistence of the virus in the body, t is time, as it is against the background of persistent human papillomavirus infection develops intraepithelial neoplasia, in 15,0-20,0% of cases leading to carcinoma in situ and invasive cancer

(14-18).

The actual problem is not only threatening the incidence of this ocassional infection, but also the difficulties associated with its early diagnosis and treatment. Standardized approaches to laboratory diagnosis verification urogenital and HPV infection are insufficiently developed. The potential risk of zlokacestvennoe cervical epithelium with long lasting (persistent) human papillomavirus infection dictates the necessity for readjustment in the detection of human papilloma virus (HPV).

To establish the factors associated persistence of human papillomavirus is also important for the scientific substantiation of the criteria for forming groups dispensary observation, optimization of the diagnostic approach and the development of differentiated tactics of patients with latent papillomavirus infection of the cervix due to 16-m and 18-m virus genotypes on the basis of medical history and laboratory criteria at different variants of the current infection.

The closest technical solution to the claimed is a method for predicting precancerous conditions of the cervix in women with human papillomavirus infection by examining biological material, determine the time the ranks of indexes, on the basis of which rely correlative index of proliferation. On the basis of the values of the last predict the degree of development of the disease (19).

This invention allows an individual determination adequate tactics sick, and the method of calculating the reciprocal of the index of proliferation is straightforward and can be enforceable in any institution of practical health care, however, this method is suitable to determine on preclinical stage only the risk of developing cervical intraepithelial neoplasia - CIN 1 and 11.

The objective of the invention is to develop a unified technique for the diagnosis of human papillomavirus infection.

The technical result of the invention consists in taking a history and comprehensive clinical and laboratory examination. As anamnestic signs take burdened cancer heredity, early sexual activity, the age of 20 years and promiscuity. As clinical signs of use anogenital warts, erosion and ectopia cervical contact bleeding, Ovuli nabotti, inflammatory diseases of the pelvic organs and the presence of intrauterine devices. As a laboratory signs take sexually transmitted infections, mystificatory, the deficit of lactobacilli, dysbiosis in agelisa, caused by conditionally pathogenic flora, bacterial vaginosis, virus-viral Association, anogenital herpes, urogenital Mycoplasma infection, urogenital replacenew infection, urogenital candidiasis urogenital chlamydial infection and infection by various HPV genotypes. Then each feature is evaluated according to a scoring system as described in the description of table 1 "Differential characteristics of transient and persistent latent papillomavirus infection", and if the patient 55-86 points diagnose persisting for human papillomavirus infection, in the presence of 0-31 points diagnose transient for human papillomavirus infection (RSI), and when 32-54 scores of patients subjected to additional tests.

To improve the diagnosis can additionally be taken into account such laboratory signs, as the average level of concentration of the oncoprotein, IFN-α, TNF-α, the average level of prolactin in the serum, the average level of interferon α and γ in the secret of the cervical canal and the average level of secretory immunoglobulin A.

The invention consists in a complex approach to diagnosis of human papillomavirus infection depending on the version of the current infection.

Acceptance as an anamnestic signs burdened ecologicallysustainable, early initiation of sexual intercourse, age 20 and promiscuity; as a clinical - anogenital warts, erosion and ectopia cervical contact bleeding, Ovuli nabotti, inflammatory diseases of the pelvic organs and the presence of intrauterine devices; laboratory infections, sexually transmitted diseases, mystificatory, deficit of lactobacilli, vaginal dysbiosis caused by conditionally pathogenic flora, bacterial vaginosis, virus-viral Association, genital herpes, genital Mycoplasma and replasment infections, urogenital candidiasis urogenital chlamydial infection and infection by various HPV genotypes is necessary and sufficient to determine the current version of The MBC.

Evaluation of each of the signs on a scoring system and diagnostics when 55-86 points persisting currents of the MBC, when 0-31 points transient currents of the MBC, and when

32-54 points additional examination of patients allows to identify the version of the persistent currents of the MBC.

Additional use as a laboratory signs of mid-level concentration of oncoprotein, IFN-α, TNF-α, the average level of prolactin in the serum, the average level of interferon α and γ in the secret of the cervical canal and the average level of secretory immunoglobulin And allows at ocnet option currents of the MBC cervix and thereby improve diagnostic accuracy.

From the analysis of scientific-technical and patent literature inventive combination of features have been identified that allows to make a conclusion on the conformity of the proposed technical solution the criteria of "novelty" and "inventive step".

The invention is carried out as follows.

At the first stage examined the epidemiological evidence. Testing of oncogenic types of HPV should be subject to sexual contacts of men with sexually transmitted infections, including viral etiology; with a pointed condylomas, bowenoid populism; giant pointed warts Buske-LJ; erythroplasia Queyras, penile cancer; patients with early age of first sexual intercourse (14-16 years); those promiscuous behavior.

Then examine laboratory signs. The latent form of human papillomavirus infection is diagnosed only by using molecular-biological methods - identification of high-risk HPVs by polymerase chain reaction (PCR) in the absence of clinical, colposcopic and cytological changes of the cervix.

Following is the definition of oncogenic genotypes of human papilloma virus in the urogenital tract, concomitant infection with human papillomavirus different genotypes (see drawing).

By showing the limits - colposcopy, staining smear on Papanicolaou (Pap-test), research performance of the immune system, including local immunity.

Is a complex bacteriological, microscopic, molecular biology (PCR) study detachable urethra, vagina and cervical canal for N.gonorrhoeae, T.vaginalis, C.trachomatis, U.urealyticum, M.hominis, M.genitalium, G.vaginalis, yeast-like fungi of the genus Candida, Herpes simplex virus I, II, Cytomegalovirus, identifying flora of the urogenital tract with regard to the number and type of microorganisms and the sensitivity of microflora to antibiotics, revealing mystificatory by sexually transmitted infections. When verification of concomitant sexually transmitted infections, and conditionally pathogenic microflora in diagnostically significant titers treatment of the patient and her sexual partner is appointed in accordance with the sensitivity to antibiotics. Related to human papillomavirus infection, sexually transmitted diseases, lengthen the treatment time and increase the risk of recurrence. When dysbiotic processes on the background of changing the pH of the vaginal environment and tissue hypoxia increases the risk of activation of viral and other infections.

The second phase will define the current version of human papillomavirus infection (transient, persistent), identifying patsie the tov risk factors for persistence of human papillomavirus by summing up and evaluation points.

Criteria persisting currents of the MBC are cases three or more allocation of human papilloma virus HPV (16/18 genotypes in cervical scrapings channel by PCR for an extended period of time when the sampling interval of 3-6 months regardless of the change of the sexual partner. Criteria transient currents of the MBC are considered to be infection with human papilloma virus HPV epithelial cells of the cervical canal in a short period of 3-6 months, when the human papillomavirus was detected once, with subsequent negative results.

Gradation characteristics are presented in order of importance and frequency of occurrence in patients with various over the MBC (table 1). The table shows the possible values of the scoring features.

Dysbiosis of the vagina caused by conditionally pathogenic microflora (Str. agalactiae, Enterococcus spp, E. coli, Proteus spp, Staph. aureus), amid a sharp decrease or absence of lactobacilli
Table 1
Differential characteristics of transient and perestroyka latent papillomavirus infection
No.SignPatients with persistent RSI [n=95]Patients with transient of the MBC [n=112]
abs%pointsabs%
Anamnestic signs
1Burdened cancer heredity4244,252421,4
2Early sexual activity (14-17 years)2829,532219,6
3The age of 20 years1212,6132,7
4Promiscuity6164,26 4842,9
Clinical signs
5Anogenital warts3031,632623,2
6Erosion and ectopia of the cervix5962,164237,5
7"Contact" bleeding5153,753632,1
8Ovuli nabotti2223,2298,0
9Inflammatory diseases of the pelvic organs16 16,821210,7
10Intrauterine devices1212,6121,8
Laboratory signs
11Of sexually transmitted infections8488,498071,4
12Mystificatory (infectious index 3 or more pathogens)63to 66.374641,1
13The deficit of lactobacilli6972,674842,9
143031,632925,9
15Bacterial vaginosis3840,042421,4
16Virus-viral Association4547,453228,6
17Anogenital herpes1717,9276,3
18Urogenital Mycoplasma infection (Mycoplasma hominis, Mycoplasma gemtalium)2223,221715,2
19Urogenital Ureaplasma infection 3031,632421,4
20Urogenital candidiasis2021,1232,7
21Urogenital chlamydial infection2627,432320,5
22Infection by various HPV genotypes(16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59) with a range of infectious index from 2 to 65254,7598,0
Additional laboratory signs
23The average concentration levels of the oncoprotein E7 HPV 16, ng/g protein 7,95±0,470,89±0,07
24IFN-α, PCG/ml in serum urovi12,7±2,926,4±3,1
25TNF-α, PCG/ml in serum24,9±6,453,8±10,9
26The average level of prolactin in serum, mIU/ml553,9±36,29321,3±26,03
27The average level of α-interferon, PCG/ml2311±500486±237
28The average level of γ-interferon, PCG/ml2379±701,51218±432
29The average reception level is Torno of immunoglobulin A (sIgA), mg/ml348,9±4,2covers 175.6±6,4

When identifying the patient of the human papilloma virus HPV after clarification of complaints and history taking, examination and clinical-laboratory examination of the patient summed up all the points in accordance with the gradation shown in table 1 signs. If there 0-31 points registered for transient HPV infection.

If patients 55-86 points is diagnosed persisting latent anogenital human papillomavirus infection and treatment that helps to reduce the risk of neoplastic processes and prevention of cervical cancer.

If there 32-54 points patients need further examination to decide on the course of human papillomavirus infection.

When doubtful a result of human papillomavirus infection use additional laboratory signs: the level of interferon (IFN-α), tumor necrosis factor (TNF-α in the serum), the level of prolactin in the serum level of α - and γ-interferon in the secret of the cervical canal, the levels of secretory immunoglobulin A (sIgA).

When identifying patients with intraepithelial neopla the iej, identified dermatological reception, it is advisable to direct them to observe a gynecologist with constant supervision by the dermatologist: at high risk of 1 times in 3 months and again in six months - at low risk. When transient option, the current human papillomavirus infection of the cervix, in the absence of manifest symptoms, you need clinical supervision in gynecological facility with periodic testing for human papillomavirus once in 3-4 months for 1.5 years.

When the latent period of human papillomavirus infection requires monitoring and regular gynecological examination in institutions (Cytology smears, fingerprints and colposcopy).

On the presented algorithm surveyed 207 patients (95 - with persistent human papillomavirus infection and 112 patients with transient over human papillomavirus (HPV) Advisory STI reception of UHF "Ural scientific research Institute of dermatology and immunology for medical technologies" (Arnieboy).

The infection rate of HPV and the nature of the flow of human papillomavirus infection largely depends on the complex influence of risk factors. The application step of the algorithm of the survey helped to identify the characteristics that accompany the persistence of the human papilloma virus is the ne, allowing way of distinguishing persistent human papillomavirus infection and treated at earlier stages.

The signs accompanying the persistence of human papillomavirus include: family history of cancer at various sites (2.1 times more often with persistent human papillomavirus infection as compared with transient during infection), inflammatory diseases of the pelvic organs in history (1.6 times), the presence of background, precancerous and benign neoplasms of the reproductive system (erosion and ectopia of the cervix) (1.7 times), the first manifestations of human papillomavirus infection in the form of genital warts (1.4 times), signs of chronic inflammation of the cervix, such as contact bleeding (1.7 times) and corking Nabatova glands (2.9 times), the use of intrauterine devices as methods of contraception (7 times), mystificatory other sexually transmitted infections (1.6 times) with the prevalence of virus-virus associations (herpes simplex virus type I-II type, cytomegalovirus) (1.7 times); the deficit of lactobacilli (1.7 times); violations of the microbiocenosis of the urogenital tract (1.2 times); bacterial vaginosis (1.9 times); anogenital herpes (2.8 times); Mycoplasma infection (1.5 times); urogenital Candide is C (7.8 times); urogenital chlamydiosis (1.3 times); the expression of the E7 oncogene of human papillomavirus (diagnostic criterion neoplastic potential risk) (10.4 times), infection of different genotypes of human papillomavirus (6.8 times), expressed the intensity of local α - and γ-interferonogenesis in cervical secretions (α-interferon 1.9 times, γ-interferon 3.8 times higher), high levels of prolactin in the serum; the increase in secretory sIgA in vaginal secretion.

Below are clinical examples of statements from the patient card.

Patient B-VA, N.G., 33 (outpatient map No. 2145). He complained of the absence of pregnancy within 2 years. When examining prenatal diagnosed with primary infertility, cystic changes of the ovaries, chronic adnexitis.

For further medical examination and treatment, the patient was referred to Arnieboy.

From the anamnesis it is known that the woman was repeatedly tested on sexually transmitted infections was identified human papillomavirus HPV (16 genotype), other sexually transmitted infections denies a history of cancer is not burdened.

Menstruation of 14 years, the cycle is broken, 25-35 days 4-7 days, painful on the first day. Sexual life is 19 years old, is married. Other sex denies. Methods of contraception does not apply. My husband is a second marriage, the first marriage and two children.

At survey: peritoneal symptoms negative, external genitals correctly formed adult female type, inguinal lymph nodes are painless, not increased, elastic. The mucous membrane of the vestibule of the vagina and urethral sponge without signs of inflammation, secretions from the genital slit no. In mirrors: cervix cylindrical shape, Zev - point, expressed contact bleeding in the collection of pathological material (5 points), from the cervical canal - transparent detachable, rear arch - rich homogeneous lucabrasi discharge with an unpleasant "fish" smell. Palpation: the uterus is not enlarged, painless, soft, mobile; ovaries on both sides - not enlarged, painless, dense. Arches free, infiltrates in the parametrium no, perianal area is not changed.

During examination: in smears from the urethra and vagina gonorrhea, Trichomonas, yeast-like fungi of the genus Candida is not detected; identified "key cells", the number of cells is not increased. "Linetest positive. In the study sosabravo material from the urethra by PCR was detected HPV 16 genotype; chlamydia, VI is the condition herpes simplex and cytomegalovirus not found. Bacteriological examination of Ureaplasma and Mycoplasma are not identified. When sowing the vaginal discharge on 5% blood agar growth of Lactobacillus spp. 103(7 points), Staphylococcus epidermidis 103, Gardnerella vaginalis 104(4 points). In the study of blood serum by ELISA revealed radiopacities Ig M in the title 1/200, radiopacities Ig G in the titer of 1/40, species-specific Ig G to Chlamydia trachomatis in the titer of 1/20, species-specific Ig And to Chlamydia trachomatis in the titer of 1/20, antibodies to lipopolysaccharide antigens (rnga) in the titer of 1/320 (3 points).

In the study of immunological parameters revealed a decrease in the relative content of T-lymphocytes, in particular T-helpers/inductors, the imbalance of immunoregulatory cells, increased levels of immunoglobulin G (leukocytes 5,4, lymphocytes 38% - 2,1, CD 3+42% is 0.86, CD 4+13% - 0,27,

CD 8+18% - 0,37, CD 16+15% - 0.31 in, CD 19+20% - 0,41, CD4+/CD8+0,72, IgA 1.6 g/l, IgM 1.5 g/l, IgG 18.0 g/l, TNF-α 1,5 PCG/ml, IFN-α 0 PCG/ml

Colposcopy showed signs of inflammation of the cervix. Cytological study of the morphological changes is not revealed. An ultrasound of the pelvic organs: signs of chronic inflammation of the ovaries on both sides (2 points), small cystic changes of the ovary.

The husband of the patient in the scrapings from the urethra identified chlamydia by PCR method.

Was diagnosed with chronic urogenital is chlamydia, bacterial vaginosis, a latent papillomavirus infection of the cervix (transient current), chronic adnexitis outside the acute stage, cystic changes of the ovary.

Used developed scoring the current version of anogenital human papillomavirus infection in conjunction aggravating signs. In this clinical case, the amount totaled 21 points (<31 points), indicating that transient for human papillomavirus infection. When re-examination of the patient after 12 and 18 months HPV 16 genotype was not detected. Cytological and colposcopic examinations - changes are not detected.

The patient M, 34 (AMB. map No. 812). Has addressed to the gynecologist for a routine examination and was sent to Arnieboy with suspected intrauterine infection.

From the anamnesis: repeatedly examined on sexually transmitted infections was twice found a Ureaplasma (3 points), herpes simplex virus type II (5 credits). Were assigned to the treatment. Cancer heredity burdened by lung cancer (5 points).

Menstruation is 12 years old, painful cycle over 26 days to 5 days. Sex life with 17 years outside of marriage (3 points). Just had 5 sexual partners (6 points). Currently divorced.

The three pregnancies ended in childbirth and two, the effect is NSTEMI abortion. History: resection of the ovaries (polycystic) (2 points), diagnostic curettage (the remains of the ovum), cervical erosion (6 points). From personal prevention of sexually transmitted infections, condoms.

At survey: peritoneal symptoms negative, external genitals correctly formed adult female type, inguinal lymph nodes are painless, not increased, elastic, mucous membrane of the vestibule of the vagina and urethral sponge without signs of inflammation, secretions from the genital slit no. In the inspection in the mirror: the cervix cylindrical shape, expressed contact bleeding in the collection of pathological material (5 points), in the posterior vaginal fornix abundant homogeneous lucabrasi discharge with an unpleasant "fish" smell.

Palpation: the uterus is not enlarged, painless, soft, movable, the right ovary is enlarged. Arches free, infiltrates in the parametrium no, perianal area is not changed.

In the reading material from the urethra and vagina gonorrhea, Trichomonas, yeast-like fungi of the genus Candida - not detected; identified "key cells", the number of cells is not increased. Minutest positive. In the study sosabravo material from the urethra by the method of the CR detected HPV 16 genotype. When the molecular genotyping of human papilloma virus detected 16, 39, 59 genotypes (5 points). Chlamydia, cytomegalovirus not found. Bacteriological examination of Ureaplasma and Mycoplasma are not identified. When planting vaginal discharge noted the growth of Gardnerella vaginalis 105(4 points), Streptococcus agalactiae 105(3 points), absence of lactobacilli (7 points).

In the study of the level of E7 oncoprotein was found to be increasing its concentration up to 10.16 ng/g protein. Was marked by elevated levels of prolactin in serum - 553,9±36,3 mIU/ml

Clinical diagnosis: Bacterial vaginosis, persistent human papillomavirus infection of the cervix, aerobic vaginitis, genital herpes (7 points).

When assessing the current version of anogenital human papillomavirus (HPV) amount of aggravating signs totaled 61 points (>56 points), indicating a persistent course of human papillomavirus infection. When re-examination of the patient the HPV 16 genotype was detected during repeated cytological and colposcopic examination revealed signs of atypia (intraepithelial neoplasia - CIN I degree).

Implementation costs of early molecular genetic screening of human papillomavirus 25% below the cost of treating patients in developing complicated forms of pupil amairani infection.

Thus, the basic principles of differential diagnosis of persistent and transient currents of human papillomavirus infection of the urogenital tract in the early stages, based on clinical and medical history, laboratory: molecular-genetic, biological, and immunological characteristics of the course of human papillomavirus infection, the factors persistence of human papillomavirus, which allowed us to develop an algorithm for stage inspection, the tactics of conducting the patient, to form a group of potential risk focal dysplastic and malignant transformation of the epithelium of the urogenital tract, early treatment in 95.8% of the patients, to optimize the prevention of the formation of persistent forms of genital human papillomavirus infection.

References

1. Rogovskaya, S. human Papillomavirus infection in women and cervical pathology: a Guide for the practitioner. - M.: GEOTAR - Media, 2005. - 144 S.

2. Adaskevich VP of sexually transmitted Diseases: Monograph // Vitebsk, 1997. - S-175.

3. Tikhonov LI Overview of STDs /STIs. - 1999. No. 1. - P.15-17.

4. Minkin G.N., Manukhin IB, Frank G. A. cervical Precancer): Aerografia, 2001. - 112 S.

5. International Agency for Research on Cancer (IARC), Working Goup / IARS monographs on the evaluation of carcinogenic risk. Human papillomavirus. IARC Sci Publ. - 1995. - Vol.64: Lyon, France.

6. Dubno CENTURIES Urogenital and HPV infection (literature review) // Russian journal of skin and venereal diseases. - 2000. No. 5. - P.50-55.

7. Beneva I.e., Prilepskaya V.N. Human papillomavirus infection and cervical pathology (literature review) // Gynaecology. - 2001. - V.3. No. 3. - P.77-81.

8. Rogovskaya, S. human Papillomavirus infection of the lower genital, clinic, diagnostics, treatment. Abstract. Diss.... d-pa honey.. Sciences. M., 2003. - 38 S.

9. Kubanov A.A., Kisin VI the Frequency and nature of background and dysplastic cervical processes in bacterial and viral infections // journal of dermatology and venereology. - 2003. No. 2. - S.43-46.

10. Kubanov A.A. Complex immunological and molecular diagnosis of human papillomavirus infection in patients and determining the formation of malignant transformation of epithelial tissues. / Abstract. Diss.... Dr. med. Sciences. - M - 2005. - 44 S.

11. Critchlow CW, L.A. Koutsky Epidemiology of human papillomavirus infection. Genital warts. Human papillomavirus infection / Ed. Mindel. London: Edward Arnold 1995; P.53-81.

12. Dillner J., Lethinen M, Bjorge T. et al. Prospective seroepidemiologic study of human papillomavirus infection as a risk factor for invasive cervical cancer // J. of National. Cancer Institute. - 1997, Sep. 3. - Vol.89, No. 17. - P.1293-1299.

13. Bekkers, R., Meijer C., L. Massuger et. al. Effects of HPV detection in population-based screening programmes for cervical cancer; a Dutch moment // Gynecol Oncol. - 2006. - Mar. - 100 (3). - P.451-454.

14. Harmsel Century, Smedts f, Kuijperset J. et al. Relationship between human apillomavirus type 16 in the cervix and intraepithelial neoplasia // Obstet Gynecol. - 1999. - Jan. 93 (1). - P.46-50.

15. Bosch F.X., Lorincz, A., Munos, M., Meijer C.J., Shah K.V. The causal relation between human papillomavirus and cervical cancer //J Clin Pathol. - 2002. - 55. - P.244-265.

16. Cuschieri, K., Whitley, M., H. Cubie Human papillomavirus type specific DNA and RNA persistence - implications for cervical disease progression and monitoring // J Med Virol. - 2004, May. - 73 (1) H. P 65-70.

17. Khan M., Castle, P., A. Lorincz et al. The elevated 10-year risk of cervical precancer and cancer in women with human papillomavirus (HPV) type 16 or 18 and the possible utility of type-specific HPV testing in clinical practice // J Natl Cancer Inst. - 2005. - 97. - P.1072-1079.

18. Schlecht N.F., Kulaga s, Robitaille J. et al. Persistent human papillomavirus infection as a predictor of cervical intraepithelial neoplasia // JAMA - 2001; 286. - P.3106-3114. The first stage of implementation of this technology is the determination of clinical and epidemiological evidence (the formation of risk groups) for in-depth examination of women in oncogenic HPV types.

19. Patent RU No. 2310197, IPC G01N 33/483, G01N 33/55, publ. 10.11.2007,

1. Method for the diagnosis of human papillomavirus infection by taking a history and comprehensive clinical and laboratory examination, characterized in that as anamnestic signs take burdened cancer heredity, early sexual activity, the age of 20 years and promiscuity; as a clinical - anogenital warts, erosion and ectopia cervical contact bleeding, Ovuli nabotti, inflammatory diseases of the pelvic organs and the presence of intrauterine device; as a laboratory infection transmitted sexually is here, mystificatory, the deficit of lactobacilli, vaginal dysbiosis caused by conditionally pathogenic flora, bacterial vaginosis, virus-viral Association, anogenital herpes, urogenital Mycoplasma infection, urogenital Ureaplasma infection, urogenital candidiasis urogenital chlamydial infection and infection with different genotypes of human papilloma virus, and then estimate each of the signs on the point system, as shown in table 1 Differential characteristics of transient and persistent latent papillomavirus infection and the patient 55-86 points diagnose persisting for human papillomavirus infection, in the presence of 0-31 points diagnose transient for human papillomavirus infection, and when 32-54 points patients subjected to additional tests.

2. The method according to claim 1, characterized in that it further as a laboratory attributes can be considered the average level of concentration of the oncoprotein, IFN-α, TNF-α, the average level of prolactin in the serum, the average level of interferon α and γ in the secret of the cervical canal and the average level of secretory immunoglobulin A.



 

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10 tbl

FIELD: medicine.

SUBSTANCE: invention concerns medicine, namely to laboratory diagnostics of human tularemia and concerns differentiation of infectious and postvaccinal antibody response at human tularemia. The essence of the invention consists that as an antigenic preparation in addition to LPS Fransicella tularensis in a dot blotting in parallel LPS Fransicella novicida is used. For this purpose LPS preparations are preliminary allocated from corresponding strains which are dissolved in the distilled water and applied on a nitrocellulose membrane in volume 1 mcl from the 1-5 mg/ml solution, with the subsequent processing by a buffering normal saline solution at pH 7.0-7.1 with 1% bull seralbumin and 0.5% twin 20 within one hour. After that the filters are washed out and incubated in the investigated serum dissolved not less than 1:100, within an hour at temperature of 37°C, then the samples are washed three times, also presence of complexes an antigen-antibody is revealed by a withstanding within 1 hour at a room temperature in a working solution of protein A, labelled with horse-radish peroxidase, with the subsequent washing up and placing in a painting solution, thus the account and an estimation of results are spent on presence of two brown maculae on a place of drawing of LPS F.tularensis and LPS F.novicida preparations which presence testifies to infectious process at the investigated patient, and at vaccinal process one maculae on a drawing place only LPS F.tularensis is observed.

EFFECT: advantage of the invention consists in simultaneous revealing infectious and postvaccinal antibody responses.

3 ex

FIELD: medicine.

SUBSTANCE: invention refers to mycoplasmoses laboratory diagnostic techniques and can be used in veterinary medicine. Method for making erythrocytic disgnosticum for indirect hemagglutination reaction (IHR) in pig's mycoplasmosis consists from fractional formalinisation of sheep's erythrocytes and sensitisation with mycoplasmosis antigen at 70°C within 30 minutes. And for sensitisation, erythrocytes are used being loaded with sensitine made of mixed mycoplasma cultures (M.hyosynoviae, M.hyorhinis and Ureaplasma sp.) taken in equal proportions and heated on water bath at 70°C within 30 minutes. Thereafter the diagnosticum is triply washed with a phosphate-buffer salt solution with PH-7.2.

EFFECT: higher specificity and activity of erythrocytic disgnosticum in indirect hemagglutination reaction (IHR) in pig's mycoplasmosis.

3 tbl

FIELD: medicine.

SUBSTANCE: method of preparing diagnostic agglutinating serum for pathogenic Yersinia strains involves hyperimmunisation of rabbits with antigen representing 0.4-0.6% formalin inactivated antigen (pYV+) of Yersinia enterocolitica My 03R strain into auricular cranial vein. Immunisation of rabbits with said antigen is fourfold in dosage as follows: 290-310 million kl/ml, 490-510 million kl/ml, 0.99-1.1 billion kl/ml and 1.9-2.1 billion kl/ml, respectively with dosage interval 6-7 days. Further the producer is examined for immunogenic properties. Serum separated from the sampled blood is preserved.

EFFECT: method ensures preparation of high-quality agglutinating serum used in yersiniosis diagnostics in animals.

3 ex

FIELD: medicine.

SUBSTANCE: invention relates to obtaining mycoplasmosis diagnostic serums for diagnosis of mycoplasmoses in pigs in indirect immunofluorescence reaction. Method includes use of antigens from three mycoplasma cultures - Mycoplasma hyosynoviae, M. hyorhinis and Ureaplasma sp.B which are used for rabbit hyperimmunisation during 21 days by divided intravenous antigen introduction every three days with triple application of immunostimulator levomisol. Antibody synthesis dynamics is studied on 7, 14 and 21 day after first antigen introduction in indirect immunofluorescence reaction (IIFR). Reaction is carried out according to conventional methodology. Four-cross system is used for reaction estimation. It has been established that on the 7-th day after first antigen introduction antibodies in IIFR were detected in low titers 1:10-1:20. On the 14-th day positive reaction in IIFR was at higher dilutions within 1:320-1:640, and on the 21-st day antibody synthesis reached maximal level 1:640-1:1280.

EFFECT: obtaining anti-mycoplasmosis serums in higher diagnostic antibody titers, reduction of hyperimmunisation terms and possibility to use obtained serums for diagnosis of mycoplasmoses in pigs in IIFR.

5 tbl

FIELD: veterinary science.

SUBSTANCE: invention refers to veterinary microbiology and biotechnology can be used for development of specific colibacillosis diagnostic aids. According to the invention the method covers producing antibody erythrocytic colibacillosis diagnosticum and provides carrier processing with antibodies to Escherichia antigens being adhesive antigens extracted from Escherichia cultures with phosphate-carbamide buffer 1.8-2.0 M of pH 7.2-7.4 at temperature 40-45°C during 25-30 min. The culture fluid containing Escherichia cell culture and phosphate-carbamide buffer are taken in mass ratio 1:0.4-0.6 respectively. The supernatant from the extraction is heated up at 65-68°C within 25-30 min. Gamma globulin fractions are used as serum antibodies for carrier processing.

EFFECT: method allows for high-quality end product due to improved sensitivity and specificity.

3 ex

FIELD: veterinary science.

SUBSTANCE: invention refers to veterinary microbiology and biotechnology, can be used for development of specific diagnostic aids. According to the invention the method covers producing antibody erythrocytic pasteurellosis diagnosticum by carrier processing with serum antibodies of Pasteurella antigens being capsular antigens extracted from Pasteurella cultures with 2.0-2.5% sodium chloride brine at 40-42°C during 30-40 min, with centrifuging the extract, separating the supernatant thereafter warmed up at 65-70°C during 15-30 min followed with sterilisation filtration. The serum antibodies for making diagnosticum are gamma globulin fraction or G or M immunoglobulins.

EFFECT: application of the method allows for high-quality end product due to improved sensitivity and specificity.

2 cl, 9 ex

FIELD: medicine.

SUBSTANCE: set of synthetic oligonucleotides includes primers 5'-CGT GTG TTG CTG GGG GAA AC-3', 5'-GCG GGT TGA GCA AGG TGT TC-3' and sample: (BHQ1)-5'-GGC GAA GCG CTT GTC C(FdT)T GGG ATT-3'P.

EFFECT: by means of PCR, set makes it possible to reliably detect DNA of causative agents in biological material of vegetable crop - Gram-negative bacterium Pseudomonas corrugata.

FIELD: medicine.

SUBSTANCE: virulent anthrax bacillus strains are grown on dense nutrient medium. Antigens of S-layer are diffused into nutrient agar and are detected with appropriate serums by creation of immunoprecipitation lines. Capsule is identified visually by morphology of colonies and with the help of light microscopy of smears colored by Rebiger. At the same time method uses non-plasmid strain B. anthracis 81/1TR and toxigenic strain B. anthracis STI.

EFFECT: developed method is an affordable method, considerably simplifies generally accepted scheme for identification of anthrax causative agent and reduces time required for analysis performance.

12 dwg, 1 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: nutrient medium contains agar-agar, lactose, mannitol, peptone, NaCl, neutral red, bromthymol blue and 0.2% solution of peat oxidate (cryall).

EFFECT: wider assortment of nutrient mediums.

2 tbl, 3 ex

FIELD: chemistry.

SUBSTANCE: atoxigenic strains of cholera vibrios 01 and 0139 serogroups are differentiated from toxigenic strains on presence of hydrolase activity. The substrate used is glycerin, which is introduced into molten Marten's agar at 2.5/100 ml of the medium with pH 7.8±0.1 and Nile blue sulphate indicator 1.5 mg/100 ml of medium, which is poured into petri dishes. Analysed broth cultures grown in a thermostat for 4 hours are deposited in volume of 20 mcl on a sector of developed agar and inoculations are incubated at 37°C for 36 to 48 hours. Strains are differentiated from their results of hydrolase activity: of the colonies grown on the medium are in form of soft semitransparent films, merging with the colour of the medium, then the analysed strain of cholera vibrio is toxigenic and epidermic. The colonies are a thick yellow or whitish deposit, then the strain is non-toxigenic haemolytic and is not epidemic.

EFFECT: method is disclosed for differentiating atoxigenic strains of cholera vibrios 01 an 0139 sergroups from toxigenic strains on hydrolase activity.

3 tbl, 3 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to microbiology. Day old agar bacterial cultures are inoculated in a single loop in a sodium chloride solution on a sector of culture medium, which contains barium ions of barium chloride, and the same culture medium without barium ions (control). Seeds are incubated at 28°C for 24 hours, after which Pseudomonas bacteria is identified from lack of growth of bacteria on the culture medium with barium ions and growth of bacteria on the same culture medium without barium ions. Presence of yellow-green fluorescence of bacterial lawn and the culture medium surrounding it indicates a group of fluorescent pseudomonade. Synthetic culture medium is used with the following content of ingredients, g/l: L-proline 1.0 to 3.0, L-sodium glutamate or L-glutamine 1.0 to 3.0, NaCl 5.0, MgCl2•6H2O 0.05, KH2PO4 0.05, K2HPO4 0.1, bacteriological agar 15.0, distilled water up to 1 litre, pH - 7.2+0.2, barium chloride supplement (BaCl2•2H2O) 6.0 to 16.0. The ingredients, except barium chloride, are dissolved while heating. The medium is boiled for 5 minutes. Part of the ready medium is poured into sterile Petri dishes (culture medium without barium ions). An amount of barium chloride calculated using a given formula is added to the remaining part of hot medium. The mixture is stirred and poured into sterile dishes (culture medium with barium ions).

EFFECT: invention increases standardness and simplifies identification of Pseudomonas bacteria.

2 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: method involves deactivation of definite VGC2 DNA sequence of Salmonella typhimurium positioned between ydhE and pykF genes or its part containing at least 50 nucleotides, or the DNA version of at least 85% identity, representing VGC2 DNA of any microbe out of Salmonella aberdeen, Salmonella gallinarum, Salmonella cubana and Salmonella typhi.

EFFECT: obtainment of microbe with reduced adaptability to specific environmental conditions.

6 cl, 12 dwg, 8 ex

FIELD: medicine.

SUBSTANCE: invention can be used for extracting bioluminescent bacteria from sea water. Growth medium consists of sea water taken in the extraction area of bioluminescent bacteria and dry nutrient agar. Medium pH is 7.2-7.4.

EFFECT: increasing bioluminescent bacteria output.

1 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: nutrient medium contains pancreatic digest of fresh-water fish, pancreatic digest of infusion broth waste, agar, sodium chloride, sodium carbonate, lithium chloride, vitamin B1, esculin, ammoniacal ferrum citrate, polymyxin B sulphate, ceftazidime, acriflavine hydrochloride and distilled water.

EFFECT: improved inhibiting and differentiating effect of the medium, reduced time for Listeria recovery.

3 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: nutrient medium contains pumpkin water, ammonium phosphate twice-substituted by (NH4)2HPO4, sodium chloride (NaCl), microbiological agar and boiled tap water.

EFFECT: extended range of nutrient mediums.

1 tbl, 4 ex

FIELD: medicine; microbiology.

SUBSTANCE: liquid nutrient medium containing L-arginine, L-cysteine, L-histidine, L-isoleucine, DL-methionine, DL-leucine, L-lysine, the L-proline, DL-threonine, L-tyrosine, L-valine, L-asparaginic acid, L-serine, glucose, sodium chloride, magnesium sulphate, thiamine, calcium pantothenate, twice-substituted sodium phosphate, monosubstituted potassium phosphate, distilled water at a parity of phases 1:1, is layered on sterile perfluorodecalin, representing the lower phase. Inoculation of F. fcularensis is performed on border of the phases. Propagation of F. tularensis in a liquid diphasic nutrient medium is carried out in static conditions during 120 h at 37°C temperature.

EFFECT: increase of an output of cells of a microorganism.

1 tbl, 8 ex

FIELD: biotechnology, microbiology.

SUBSTANCE: method for preparing liquid lactobacterin involves regeneration, culturing passages of lyophilized culture and culturing ferment of lactobacilli in liquid lyophilized nutrient medium containing dry defatted milk enzymatic hydrolyzate with the content of amine nitrogen 1 485 mg%, 30.0 ± 3.0 g/l; yeast concentrated autolyzate, 110.0 ± 10 g/l; food agar, 0.8 g/l, and distilled water, up to 1 l. Culturing ferment is carried out up to accumulation of biomass of lactobacilli 109-1010 CFU/ml. Then 10-30% of supernatant liquid is removed from the ready product and replaced it with equal volume of fresh nutrient medium. Invention provides simplifying technology in preparing liquid lactobacterin and to elevate the storage period of viable lactobacilli. Invention can be used in producing probiotic preparations.

EFFECT: improved preparing method.

1 tbl, 3 ex

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