EA025042B1 - Method for differential diagnostics of vaginal microflora balance disorders - Google Patents

Abstract

The invention relates to medicine, particularly laboratory diagnostics, and may be applied to differential diagnostics of vaginal microflora balance disorders. Vaginal smear from a patient is taken, and total bacterial DNA concentration, Gardnerella vaginalis DNA concentration, Atopobium vaginae DNA concentration and Lactobacillus spp. DNA concentration are determined via nuclear acids amplification procedure. Based on results obtained, concentration ratio coefficients are calculated: CR1=lg[DNA Lac]-lg([DNA Gv]+[DNA Av]), CR2=lg[DNA Bac]-lg[DNA Lac] and CR3=lg[DNA Bac]-lg([DNA Gv]+[DNA Av]). When lg[DNA Bac]≥5, CR1>1 and CR2≤1, no bacterial vaginosis is stated, when lg[DNA Bac]≥5, 0.5≤CR1≤1, CR2≤1 and CR3≤2, intermediate microflora condition is stated, when lg[DNA Bac]≥5, CR1<0.5 and CR3≤2, bacterial vaginosis is stated, and when lg[DNA Bac]<5, amount of bacteria is considered insufficient for the analysis. When bacterial vaginosis is absent and 5≤lg[DNA Bac]<6, decreased grade of bacterial growth in vagina is stated. Additionally, Enterobacteriaceae DNA concentration, Staphylococcus spp. DNA concentration and Streptococcus spp. DNA concentration are determined in vaginal smear via nuclear acid amplification procedure. When lg[DNA Bac]≥6, ([DNA Ent]+[DNA Staph]+[DNA Strep])≤[DNA Lac] and bacterial vaginosis is absent, conclusion is "vaginal microflora balance disorders not detected", when lg([DNA Gv]+[DNA Av])-lg([DNA Ent]+[DNA Staph]+[DNA Strep])≤1, bacterial vaginosis diagnosis is specified as "bacterial vaginosis combined with increased amount of aerobian microflora", and when lg[DNA Bac]≥5, ([DNA Ent]+[DNA Staph]+[DNA Strep])>[DNA Lac], CR2>1 and CR3>2, prevalence of aerobian microflora is stated. When local inflammatory reaction is observed combined with increased amount or prevalence of aerobian microflora, aerobian vaginitis is stated. Invention provides a tool for differential diagnostics of vaginal microflora balance disorders with high diagnostic sensitivity and specificity along with minimum consumption of material resources and time.

Description

The invention relates to medicine, more specifically to laboratory diagnostics, and can be used for differential diagnosis of imbalances in the vaginal microflora.

State of the art

Infectious diseases of the urogenital tract in women occupy the first place in the structure of gynecological pathology [Oopbegk O.O.O. Ieyi Shoi apb s1akk1ysayop o £ aiogta1 wadta1 Log. Century! Rgas !. Kek. Bn. Ok! E !. Oupaeso 1. 2007 1ip; 21 (3): 355-373. Yer 2007 Arg 16].

The causative agents of infectious diseases of the urogenital tract can be not only obligate pathogenic microorganisms (No. 1kkeya dopoggoeae, Syatufa sapiotaik, Musoracta deptum, TpsNotopacum uadshaq, etc.), but under certain conditions they are also normally pathogenic organisms.

As part of the vaginal microflora of healthy women of reproductive age is dominated by lactobacilli (Lactobacillus, Lac oasShik CRE, from the Latin kreya1ek -!.. Types), such as aLoasShik spkraShk, aLoasShik shegk, aLoasShik daShpagit, Lac oasShik dakkegk Lac oasShik uadshak, Lac oasShik!!! ] epkesh1, Laskyas Shik ashboy1ik, bas! obasShik p1ai1agit [Ed. Seagulls V.K. Infections in Obstetrics and Gynecology: A Practical Guide. Donetsk, LLC Almateo, 2006; Byshuacap 8. apb N. Rgebysk Ό.Ν. Thi Nitap Wadsha1 Vas1epa1 Vu1a apb Vas1epa1 Wadshoyk. 1p1egb1ks1r1shagu Regkresyuek op Shesyoyik EReakek, Uoite 2008, Agys1e GO 750479].

Lactobacilli, which make up the normoflora of the vagina, protect it from the multiplication of pathogenic and conditionally pathogenic bacteria. The protective factors of lactobacilli include the formation of lactic acid, which leads to the maintenance of an acidic environment of a healthy vagina, the formation of hydrogen peroxide and bacteriocins, high adhesive ability and immunostimulating properties, which together provide the colonization resistance of vaginal microbiocenosis [Ed. Seagulls V.K. Infections in Obstetrics and Gynecology: A Practical Guide. Donetsk, LLC Almateo, 2006].

In addition to lactobacilli, the following bacteria can be present in variable but insignificant amounts in the composition of the vaginal microflora: ., RikoLaileit krr., Lepyuhr krr., Satru1oLaeg krr., EeyeLaLeasee krr. (ExeCeCr., KlEbk1e11a krr., Ep1egoac! Ekr., Rgo! Eik krr.), Ashe! OeLeg kr. ., Ejegosossik krr., 81ger! Ossossik krr., RerYusossik krr., RerYukLerYusossik krr., Ueb1ope11a krr., Musor1acta botsch, Igearakta krr. (Igear1akta raguit, Igear1akta igee1uysit), as well as mushrooms Sapb1ba krr. (Sapb1ba a1Ysapk, Sapb1ba d1abga! A, Sapb | ba kalek Sapb | ba ragarkboyk, Sapb1ba loruybk), Asypotusek krr. et al. [Ed. Seagulls V.K. Infections in Obstetrics and Gynecology: A Practical Guide. Donetsk, LLC Almateo, 2006].

Adverse endogenous and exogenous factors (hormonal disorders, the use of antibiotics, oral and intrauterine contraceptives, decreased immunity, past inflammatory diseases, etc.) can lead to imbalances in the vaginal microflora, i.e. reducing the content of lactobacilli and replacing them with opportunistic microorganisms. Two main types of such disorders are known: bacterial vaginosis and aerobic vaginitis.

Bacterial vaginosis is a clinical syndrome caused by the replacement of lactobacilli by the vaginal microflora with conditionally pathogenic anaerobic and facultative anaerobic bacteria. [Ed. Savelyeva G.M., Breusenko V.G. Gynecology: Textbook. Moscow, GEOTARMED, 2004]. Bacterial vaginosis is a polymicrobial syndrome with an extremely wide range of detectable opportunistic microorganisms, numbering hundreds of species. A reliable connection with bacterial vaginosis has been demonstrated for representatives of the eight bacterial genes Ogbpepea, Aloporhon, Medacrea, Eddebie11a, Aegosossik, Ep! O! R1c1a / 8pea! B1a, Prheuo! E11a, Parbie. e! a1. Mo1e1aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa! Navy Oepotyuk. 2010 8er 7; 11: 488].

Experts also identify an intermediate type of microflora, in which there is no complete (or almost complete) replacement of lactobacilli by conditionally pathogenic anaerobic and facultative anaerobic bacteria, however, the number of the latter is significantly increased compared to the norm. An intermediate type of microflora can be considered as a transition from normal to bacterial vaginosis (just getting sick) or vice versa (restoring microflora) [No Dep! K.R. e! a1. Ь а Ь о о п п п п Л Л Л Л еп еп Л Л а а а б б б б б б!!!! А а а а а а 1 1 1 1 1 оп. 1. Cn. Hugo 1. 1991 Peb; 29 (2): 297-301].

Aerobic vaginitis is an imbalance in the vaginal microflora that is different from bacterial vaginosis. In aerobic vaginitis, lactobacilli of the vaginal microflora are replaced by conditionally pathogenic aerobic microorganisms (Epilogue Leucase crp. Ossossian krr., including Ogoyr B 8! ger! ossossik, he is 8Ler! ossossik ada1asyaye, Ogoyr A 81ger! ossossik, Eyegosossik krr.). In contrast to bacterial vaginosis, which is not inflammatory in nature, aerobic vaginitis is characterized by the development of a local inflammatory response and immune response [ΌοηάβΓδ O.O.O. e (a1. Όβίΐηί! ίοη οί а 1urs οί аЬпогта1 уадта1 yoga Ша! ίδ ΟίδΙίηοί Goth bac1sg1a1 ναβίηοδίδ: aegus wadt. VUS. 2002, 1ap; 109 (1): 34 (34).

Imbalances in the vaginal microflora can be accompanied by clinical manifestations (pathological discharge with an unpleasant odor, itching, burning, dyspareunia), but they can also be asymptomatic. An asymptomatic course is especially dangerous, since in the absence of adequate therapy, it is possible to develop such formidable consequences for the health of a woman and her offspring as inflammatory diseases of the pelvic organs, premature birth, the birth of children with low body weight, inflammatory complications of pregnancy and childbirth, and increased susceptibility to infections sexually transmitted [Ed. Savelyeva G.M., Breusenko V.G. Gynecology: Textbook. Moscow, GEOTARMED, 2004].

In this regard, it is difficult to overestimate the importance of reliable and effective diagnosis (including differential) of imbalance in the vaginal microflora, which allows you to timely determine the choice of suitable therapy. The difficulties of this diagnosis are caused, firstly, by the non-specificity of the corresponding symptoms, and in some cases by their absence, secondly, by the huge variety of opportunistic microflora species that have different properties, including different sensitivity to antibiotics, and thirdly, conditionally the pathogenic nature of pathogens, which determines the limited diagnostic value of their qualitative determination and requires only a quantitative approach.

Existing diagnostic methods do not fully cope with these difficulties.

Classical laboratory diagnostic methods based on the cultivation of microorganisms on nutrient media are not suitable for identifying uncultivated and difficult to cultivate conditionally pathogenic microorganisms, among which are microorganisms associated with bacterial vaginosis. For cultivated microorganisms, this method is also not without significant drawbacks, including the difficulties of standardization and quantification, long cultivation periods.

Amsel’s criteria traditionally used for the diagnosis of bacterial vaginosis have a low diagnostic sensitivity [§N.A. e! a1. ISHNU ίί х х 1 сг 1 сг а а а,, ид ид δ δ δ ΐΐ ΐΐ ΐΐ Юг Юг Юг Юг Юг Юг Юг Юг ίΐ ίΐ ίΐ ίΐ. Ν. Mutor ^ that Ιιοιηίηίδ. AY KASGOBASPN. ^ δρρ. Γογ άίη§ηοδίδ οΓ bac1eya1 νηφηοδίδ ίη Pitan ^ ttiηοάais ^ eηсу νίιπδ-ίηί € ^ ά \ νοηκη. 1. C1. Hugo 1. 2005 §per; 43 (9): 4607-4612] and do not provide information on the qualitative and quantitative composition of microflora, which can lead to errors in the choice of therapy.

A method for diagnosing bacterial vaginosis, based on microscopy of Gram-stained preparations with the calculation of bacterial morphotypes and the use of Nugent's criteria, is quite accurate, but it does not lack some share of subjectivity and is very laborious, which significantly reduces the laboratory capacity. In addition, some species of conditionally pathogenic microflora that are significant for the diagnosis of bacterial vaginosis, in particular ΑίοροΝιιιπ νад ^ ηае, do not have specific microscopic features and are mistaken for other species [БсПМегй Α. e! a1. Ϊ́Ό1ιΐΌ \ νίη§ (Not Is Gegh (Пе άίηβηοδίδ οί \ 'adta1 ^ 3)? Ηηη С1т МюгоЬю1 АайтисгоЬ. 2006 Re 17; 5: 4].

A method for diagnosing aerobic vaginitis based on microscopy of native drugs with an assessment of the lactobacillary degree, morphotypes of concomitant microflora and counting leukocytes and parabasal epithelial cells, is also laborious and not without some share of subjectivity | Οοηώ; Γδ С. С. С. e! a1. □ ePtNu! '! οί а! ure οί аЬгугта1 \ 'ads1 Ποπι! On! ίδ άίδίίηΟ Ggot асac1eya1 νηβίηοδίδ: aegis νΗβίηίΙίδ. VGOS. 2002 1ash 109 (1): 34-43].

Modern molecular biological methods based on the amplification of nucleic acids have many advantages over traditional diagnostic methods. The most widely used method based on the polymerase chain reaction (PCR). PCR analysis can be performed in both qualitative and quantitative formats.

PCR allows you to quickly identify, with high sensitivity and specificity, both cultivated and uncultured microorganisms in any biological sample. Quantitative PCR allows you to objectively and reliably determine the number of microorganisms. The ability to standardize and automate analysis is an additional benefit.

A known method of studying the biocenosis of the urogenital tract in women by real-time PCR and a set of reagents for its implementation FEMOFLOR 16. The total number of bacteria (total bacterial mass, MBP) in the sample, the number of lactobacilli and the number of 22 conditionally pathogenic microorganisms are determined in 16 tubes / groups of microorganisms: 16 species / genera of anaerobic bacteria (Cacterium bacillus ηa1 ^ δ / P ^ eνο! e11a L ^ ν ^ a / Pο ^ ρпу ^ οтοηаδ δρρ., Ейбас! ешп δρρ., Бте-Ийа δρρ. ! ο! ^^ Н ^ а δρρ. / ΡиδοЬас! е ^^ и δρρ., Ме ^ рПега δρρ. / Vе ^ 1οηе11а δρρ ./^^ а1 ^ δ! е ^ δρρ ., ^ acNηοbas! е ^^ um δρρ. / С1οδί ^^ ά ^ it δρρ., ΜοΝΗιικιΐδ δρρ. / Сο ^ унееас! е ^^ um δρρ., Рер ^ й ^ Ш ^ сс ^ δρρ., Α! οροЬ ^ um νηφηκΕ is one family and two genera of aerobic bacteria (Eeye, ейейейасасасаее,,, ЕерЮЮЮ ^ сс ^ ^ δ δ δρρ., BorNu ^^ ss ^ δρρ.), Mutor та No ^ η ^ δ. Igearарa δρρ., С-пЫй-δρρ. The method includes an algorithm

- 2 025042 the formation of a laboratory report on the basis of an analysis of the relative (in% of MBP) quantities of detected bacteria [FEMOFLOR. The study of the biocenosis of the urogenital tract in women by PCR with the detection of results in real time. Toolkit for laboratory assistants. FSBI SSC Institute of Immunology FMBA of Russia. ZAO NPF DNA-Technology].

However, this approach has several disadvantages. The diagnosticum includes a number of representatives of opportunistic microflora, the diagnostic value of which is not confirmed by published research results. Various researchers found a large number of various opportunistic microorganisms in the vagina of women with bacterial vaginosis, but the mere fact of such detection is not enough for the effective use of microorganisms as quantitative diagnostic markers of the disease [Mepagb TR. e1 a1. Mauciociag sschapbPsabop oG Oagbpege11a wadia Pyat apb Ayurobt wadtae 1oobya 1o rgeb1s1 bac1eg1a1 wadshoya. С1т 1пГес1 Όίδ. 2008 1 and 1. one; 47 (1): 33-43]. The inclusion of invalid markers in the study unnecessarily increases the complexity of the analysis and the complexity of its interpretation.

The issued laboratory findings (absolute / relative normocenosis, severe / moderate aerobic / anaerobic / mixed dysbiosis) do not indicate known nosologies. In addition, the authors of the present invention do not know the published data on the validation of this method, including a quantitative algorithm for generating a conclusion, regarding standard diagnostic methods for the corresponding nosology (bacterial vaginosis, aerobic vaginitis).

The closest in technical essence to the present invention is a method for the diagnosis of bacterial vaginosis, described in the international application for the invention νθ 2011/068679 ].

The method includes the quantitative determination of one or more types of lactobacilli and two or more types of opportunistic microorganisms in a biological sample and calculation based on them of a diagnostic indicator. With its value less than 0.2, bacterial vaginosis is diagnosed, with a value of 0.2 to 4.99, an intermediate type of vaginal microflora is diagnosed, and with a value of 5 or more, the normal state of vaginal microflora is diagnosed. The diagnostic indicator is the ratio of the sum of the logarithms of the concentrations of lactobacilli to the sum of the logarithms of the concentrations of opportunistic bacteria or the ratio of the logarithm of the sum of concentrations of lactobacilli to the logarithm of the sum of the concentrations of opportunistic bacteria. Conditionally pathogenic bacteria are selected from the group Auroptum uaschpae. Medayar Yarr., Oagbpege 11a Wadsha Pya.

This diagnostic method is validated against Nugent’s criteria, however, its sensitivity and specificity are low: 57 and 60%, respectively (for different options for calculating a diagnostic indicator, the values of sensitivity and specificity differ, the best characteristics are given here).

In addition, the method has another significant drawback: it distinguishes only three states of vaginal microflora: bacterial vaginosis, an intermediate type of vaginal microflora and the normal state of vaginal microflora. Violations of the balance of vaginal microflora other than bacterial vaginosis and an intermediate type of vaginal microflora (for example, the predominance of aerobic microflora, aerobic vaginitis), the method mistakenly refers to the intermediate type of vaginal microflora.

The lack of determination of the total number of bacteria in the sample does not allow us to assess the adequacy of taking a biological sample, which may lead to errors in the determination of the diagnostic indicator; it does not allow to distinguish the imbalance of the vaginal microflora of a different (non-anaerobic) genesis, as well as the state of reduction of bacterial contamination of the vagina.

SUMMARY OF THE INVENTION

The objectives of the present invention are:

creating a reliable and effective method for differential diagnosis of imbalances in the vaginal microflora, based on the quantitative determination of bacterial DNA of normal and conditionally pathogenic microflora;

increasing diagnostic sensitivity and specificity;

minimization of material and time resources spent for diagnostics through the optimal choice of bacteria-markers of vaginal microflora imbalance, i.e. selection of markers necessary and sufficient for sensitive and specific diagnostics;

improving the reliability of diagnosis using the principle of quantitative assessment of bacteria of normal and conditionally pathogenic microflora, not in absolute terms, but in relation to the total number of bacteria in the sample, which makes it possible to level the significant spread in determining the absolute concentration of microorganisms associated with the quality of taking biological material;

development of an expert algorithm for interpreting the results of quantitative analysis;

method validation, including an algorithm;

- 3 025042 expansion of the arsenal of modern laboratory diagnostic methods.

In the framework of the present invention, an analysis of the world scientific literature was carried out with the aim of optimal selection of diagnostic markers of bacterial vaginosis, i.e. the choice of such conditionally pathogenic microorganisms, the quantitative determination of which is necessary and sufficient for a sensitive and specific diagnosis of this condition.

Oagypegeia urpike is determined in 99% of women with bacterial vaginosis [Vaigayate C.8. c1 a1. Thier Akkoshayop OG Ayurbyt Uadtay Apy Oagipege 11a Uadtayk \ νί11ι bas1sg1a1 uadtopk apy gesiggepse ayeg oga1 te1goYyya / o1e Legaru. 1. 1pGes (. Όίκ. 2006 § 15; 194 (6): 828-836]. This type of bacteria, unlike other species associated with bacterial vaginosis, combines three virulence factors: the ability to adhere to human epithelial cells, biofilms and cytotoxicity Reb; 156 (P4 2): 392-399]. According to the latest data from Oagipege 11a, wadtike is etiologically m agent of bacterial vaginosis [Zsjetie TK. e (a1. Ko1e oG Oagypegeya wadtayk sh (Not RaLodepeyk oG Vas (ua1 Wadshoyyk: A Sopcer (u1 Moie1. 1. 1pGes (. .κ. 2014 Mag. 31. ]. Metronidazole is the standard therapy for bacterial vaginosis, while the discovery of other representatives of conditionally pathogenic microflora does not change the tactics of treating bacterial vaginosis. The exception is A (anoxidum, for which resistance to metronidazole has been demonstrated [Ie Vaskeg E. e (a1. Apyuys kikseryyu oG A (orst uadtay. IUD! NGes (Mar. 2006 Mag. 16; 6:51]. Accordingly, the detection of this type of bacteria in significant quantities changes the tactics of therapy.

Thus, from the entire variety of opportunistic microflora species associated with bacterial vaginosis, two species were selected as quantitative diagnostic markers of the disease: Oagypegea uadtayk and A (aryobacterium.

In order to optimally select diagnostic markers of aerobic vaginitis, literature data were analyzed and a number of experiments were carried out.

The following four genera / families of pathogens of aerobic vaginitis are known: Ep (ebac (euacae (representatives of Exxeus soy, K1ebk1e11a krr., Prgo (eik krr.), 8 (apyuossossic krr. . (representatives of Ogoyr B § (ger (ossossik, aka 8 (ger (ossossik adaassaye, Ogoyr A § (ger (ossossik)), EEgosossik krr. [Iopyegk O.O.O.e (a1. Ieyyyop oG a (ur bogta1 uadta1 yoga (ba (1k baaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa ...)

The informativity of the main groups of aerobic microorganisms at the genus / family level was studied experimentally (En (efbac (eufoss, 8 (apfu1ossosscr., 8 (ger (ossossicus cfr., Ebossossiccrr.) 8 (ger (ossossic adaaciae). The unsuitability of EEgossossik krr. As a quantitative diagnostic marker was demonstrated and low sensitivity (48%) of the aggregate of certain types of aerobic microorganisms for the diagnosis of aerobic vaginitis was established.

Thus, from the known representatives of opportunistic microflora associated with aerobic vaginitis, the following three groups of aerobic microorganisms at the level of the genera / families were selected as quantitative diagnostic markers of the disease: EEgobac (euacaea, 8 (apu1ossosscr., 8 (ger (ossossic krr.

The objectives of the present invention have been solved by creating a method for differential diagnosis of imbalances in the vaginal microflora, including determining the concentration of DNA of all bacteria, the concentration of DNA of the bacterium Oagypegeya wadtike, the concentration of DNA of the bacterium A. that based on the data obtained, the coefficients of the ratios of the concentrations are calculated:

KC1 = 1d [DNA Bas] - 1d ([DNA Oy] + [DNA Au]),

KC2 = 1d [DNA of you] - 1d [DNA of Bas],

KC3 = 1d [DNA of You] - 1d ([DNA of Au] + [DNA of Au]), where [DNA of Bas] is the concentration of DNA of bacteria BaObacia krr. in GE / ml, [DNA Oy] - DNA concentration of the bacterium Oagepegeia wadtike in GE / ml, [DNA Au] - DNA concentration of the bacterium A (ooYuid uadtae in GE / ml, [DNA of you] - DNA concentration of all bacteria in GE / ml , with 1d [DNA of you]> 5, KC1> 1 and KC2 <1, the absence of bacterial vaginosis is noted, with 1d [DNA of you]> 5, 0.5 <KC1 <1, KC2 <1 and KC3 <2, an intermediate state of microflora is observed , with 1d [DNA of you]> 5, KC1 <0.5 and KC3 <2 diagnose bacterial vaginosis, and with 1d [DNA of you] <5, it is ascertained that the number of bacteria is insufficient for analysis.

In the particular case of the method according to the invention, it is characterized in that in the absence of bacterial vaginosis and 5 <1d [DNA of you] <6, a decrease in the degree of bacterial contamination of the vagina is noted.

In the particular case of execution of the method according to the invention is characterized in that it further determines the concentration of DNA of Epilocobacillus passas. bacterial DNA concentration §Garyu1ossossi 5 VRR, bacterial DNA concentration ZGgerGossoss VRR. in the sample by the method of amplification of nucleic acids, with 1d [DNA of you]> 6 and ([DNA Εηί] + [DNA of 81 ayr] + [DNA of 8yr]) <[DNA of bac], where [DNA of асηί] is the concentration of the DNA of Epiloeuxepaseae in GE / ml, [DNA §Gary] - the concentration of DNA of bacteria §Garyu1ossossi 5 VRR. in GE / ml, [DNA §Gger] - DNA concentration of bacteria ZGgerGossoss vrr. in GE / ml, and in the absence of bacterial vaginosis, they conclude that there are no violations of the balance of vaginal microflora.

In the particular case of the implementation of the method according to the invention is characterized in that it further determines the concentration of DNA of Epigotosacrilaceae, the concentration of bacterial DNA §Garyuossoss5 vr., The concentration of bacterial DNA ZggerGossoss vrr. in the sample by the method of amplification of nucleic acids, at 1d ([DNA Ον] + [DNA --ν]) - 1d ([DNA Εηί] + [DNA 81 ar] + [DNA 8pyr]) <1, where [DNA --ηί] is the concentration of the DNA Ep1goBac1epaceae in GE / ml, [DNA §1ary] - the concentration of DNA of bacteria §1aryu1ossss VRR. in GE / ml, [DNA §1ger] - the concentration of DNA of bacteria §1ger1ossiviv VRP. in GE / ml, the diagnosis of bacterial vaginosis is specified as bacterial vaginosis in combination with an increased content of aerobic microflora.

In the particular case of performing the method according to the invention, it is characterized in that in case of bacterial vaginosis in combination with an increased content of aerobic microflora, the presence of clinical signs of inflammation in the patient and the ratio of polymorphonuclear leukocytes: epithelial cells during microscopy of the sample and in the presence of clinical signs of inflammation in the patient are additionally determined the ratio of polymorphonuclear leukocytes: epithelial cells more than 1 diagnose bacterial vaginosis in combination with aerobic vaginitis.

In the particular case of the implementation of the method according to the invention is characterized in that it further determines the concentration of DNA Εηίе ^ о осίе ^^ асеее, the concentration of bacterial DNA §1арюуossossing time, the concentration of bacterial DNA §1ger1, saving time. in the sample by the method of amplification of nucleic acids, at 1d [DNA of you]> 5, ([DNA Εηί] + [DNA of 81 ar]] + [DNA of 81 ger])> [DNA of bac], KC2> 1 and KC3> 2, where [DNA Εηί ] is the concentration of DNA ίηίе ^ оБасίе ^^ асее in GE / ml, [DNA 81ArH | - the concentration of bacterial DNA 81arNu1ossiviv VRP. in GE / ml, [DNA δί ger] is the concentration of DNA of bacteria Engerususiviv rr. in GE / ml, ascertain the predominance of aerobic microflora.

In the particular case of performing the method according to the invention, it is characterized by the fact that, with the predominance of aerobic microflora, the presence of clinical signs of inflammation in the patient and the ratio of polymorphonuclear leukocytes: epithelial cells during microscopy of the sample and in the presence of clinical signs of inflammation in the patient and the ratio of polymorphonuclear leukocytes are additionally determined: epithelial cells more than 1 diagnose aerobic vaginitis.

In the particular case of carrying out the method according to the invention, it is characterized in that when ascertaining the intermediate state of microflora, the pH of the patient’s discharge and the presence of complaints of pathological discharge in the patient are additionally determined, and at pH <4.5 and the absence of complaints of pathological discharge, the patient is recommended to repeat the analysis in the next menstrual cycle and at pH> 4.5 and the presence of complaints of pathological discharge, the patient is diagnosed with bacterial vaginosis.

In the particular case of performing the method according to the invention is characterized in that the sample is a vaginal smear.

In the particular case of performing the method according to the invention is characterized in that the method of amplification of nucleic acids is a method of quantitative polymerase chain reaction.

In the particular case of performing the method according to the invention is characterized in that the method of quantitative polymerase chain reaction is a polymerase chain reaction method with hybridization-fluorescence detection in real time.

In the particular case of performing the method according to the invention is characterized in that the polymerase chain reaction is a multiplex polymerase chain reaction.

The invention allows reliable and efficient differential diagnosis of imbalance in the vaginal microflora. When implementing the method according to the invention, an increase in sensitivity to 98.5% and specificity to 97.4% is achieved for the diagnosis of bacterial vaginosis with minimal material and time resources.

The created invention can be used both for primary diagnosis, confirmation or refutation of previously diagnosed and prescribed therapy, and for subsequent monitoring of treatment effectiveness and monitoring of cure criteria.

- 5 025042

List of figures

FIG. 1. The distribution of patients by the total number of bacteria in the sample (in 1e).

FIG. 2. Concentrations of aerobic microorganisms in patients with aerobic vaginitis.

Information confirming the possibility of carrying out the invention

The study included 274 patients of reproductive age who applied to the Institute of Obstetrics and Gynecology. BEFORE. Ott Russian Academy of Medical Sciences. The average age of the patients was 30 ± 5.7 years. Each patient was examined using both clinical and laboratory diagnostic methods to differentiate various imbalances in the vaginal microflora: bacterial vaginosis, intermediate microflora, bacterial vaginosis in combination with aerobic vaginitis, aerobic vaginitis.

The first stage of the development of a method for the differential diagnosis of imbalances in the vaginal microflora included:

1) determination of Amsel’s criteria (the presence of specific vaginal discharge, an increase in pH> 4.5, a positive amine test, the presence of key cells during bacterioscopy);

2) Gram stain microscopy with an assessment of Newgent scores (0-3 points - normal; 4-6 points - intermediate microflora type; 7-10 points - bacterial vaginosis);

3) determination of the concentration of DNA of all bacteria, the concentration of DNA of the bacterium Sagbpege 11a pars, the concentration of DNA of the bacterium Lactobacillus, the concentration of DNA of bacteria Bacillas spp. 8 pp. in a vaginal smear by nucleic acid amplification - multiplex polymerase chain reaction (PCR) with hybridization-fluorescence detection in real time.

To conduct a multiplex PCR study, each patient received a vaginal sample (vaginal smear) in a transport medium with a mucolytic (TSM, FBSI Central Research Institute of Epidemiology of Rospotrebnadzor, Moscow, Cat. No. 953) with a volume of 500 μl. Next, nucleic acids were extracted using a set of reagents for the extraction of DNA from AmpliPrim DNA-Sorb-AM clinical material (Federal State Institution of Research Institute of Epidemiology of Rospotrebnadzor, Moscow, cat. No. 102-22). For further amplification, 10 μl of the obtained eluate containing DNA of the studied microorganisms was used (the volume of the obtained eluate after extraction was 100 μl).

During the preparation of the reaction mixture, a 100-fold TE buffer was used to obtain the necessary concentration of primers, probes, and deoxyribonucleoside triphosphates (6ΝΤΡ). The composition of the reaction mixture is given in table. one.

Table 1

The composition of the reaction mixture (PCR mixture-1)

Title Sequence Fluo rofort Concentration, pmol / μl Primers Ονί 8С8-АТА-88С-1ТГ-ТСС-Т ^ -Т8Т-АТС-АСТ 5,0 Ον-Α3 STS-SSS-ATT-S88-AAA-TSS-TSE-8AT-SA 5,0 ΑΤν-ί eST-SAA-SSS-STA-TSS-8ST-SST 6.0 ΑΤν-az TA§-C§8-SAS-88A-AA _ё -TAT-AAT-STS-S 6.0 BR-5P1 T _g T AAC T§A _S-C-C T8A- £ _{8 g} TC--AA 1,5 Bp-azp A8A-88S-88A-AAS-STS-SSA-ASA-ST 1,5 51631E CAC-ATT-8§8-AST- £ A £ -ACA-Shё-SSS-A 0.6 81652M C _{g C-ACT-8-det} -ast oA8-ATA-C _Yog -SSS-A 0.6 31683P SAS-ATT-8ba-AST-8A§-ASA-S8-TCC-A 0.6 A163 S ₈ T-ATT-ACC-8C ₈ -8CT-8CT-88C-AC 1.8 Probes Ον-Ζ (Rat) -SSS-AST-S8§-SAe-STS-SSS-§A8-8CA-T- (VNf1) Rush 0.8 ΑΤν-ΐΒΙ (BWC) -SSA-A8T-§TA-8S8-8T8-AAA-C £ T _g - (VIK 1) K6O 1,5 Bp-ρη-ΚΒ (Koch) -STS-ATS-8TT-TAS-§8S-A1§-§AS-TAS-Sa8- (VfN2) Koch 1,5 ΒΥΖ-Ογ5 (Su5) -AST-SST-As§-88A-88S-A8S-A8- (KTf2) Su5 4.0 Deoxyribonucleoside Triphosphates άΝΤΡ (4.4 shM) 5,0

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To obtain the reaction mixture, 10 μl of PCR-mixture-1 and 5 μl of PCR-mixture-2 gsb were added to the test tube (FBSI Central Research Institute of Epidemiology of Rospotrebnadzor, Moscow, Cat. No. 863).

Then, 15 μl of the reaction mixture and 10 μl of DNA eluate were added to each amplification tube. In addition, calibrators were used in each formulation for the quantitative analysis of microorganisms (10 μl of calibrators were added to the corresponding tubes). Each formulation involved 2 calibrators with a concentration of 10 ⁵ and 10 ³ copies / reaction.

The composition of the calibrators (PC1 and PC2).

Calibrators include two preparations cloned into phage λ: a concentrated solution, a positive control sample (CRP) of Oagbpege11a uaschpaPh and KR PKO bac1oacaci5 5pp. . For dilution of the initial RS, a DNA buffer with po1u (A) is used. The final concentration of each preparation for the PC1 calibrator is 1-10 ⁵ copies / reaction; for the PC2 calibrator - 1-10 ³ copies / reaction.

Amplification was carried out on a Co1ot-Oeie O device (ΟΙΑΟΕΝ, 1011132).

Amplification program:

Stage Temperature ° C Time Measurement fluorescence Qty cycles But also/ retention temperature 95 15 minutes - one 1 Susnp§ / Citation 95 5 s - 5 60 20 s - 72 15s - 2 Susnp§ / Cycling 95 5 s - 40 60 20 s RAM, UE, KOH, Su5 72 15s

Next, a comparison was made of the results obtained by three methods for diagnosing bacterial vaginosis. Due to the fact that both the Amsel criteria and the assessment using Newgent scores imply a certain amount of subjectivity, both methods were used as an expanded gold standard. Assessment of the diagnostic characteristics of the method according to the invention was carried out on those samples for which the conclusion on the Amsel criteria and Nugent scores coincided.

The results of the study of samples from 274 patients are given in table. 2. For each sample, the following data:

Assessment of the state of vaginal microflora according to Amsel’s criteria - bacterial vaginosis (BV) or the absence of bacterial vaginosis (OBV);

Assessment of the state of vaginal microflora by Nugent scores - bacterial vaginosis (BV), an intermediate type of microflora (PT) or the absence of bacterial vaginosis (OBV);

bacterial concentrations, expressed in units of GE / ml, determined by the real-time multiplex PCR method: [DNA Ον] - DNA concentration of the bacterium Oagyiege11a να ^ ίηαΐίδ, [DNA Αν] - DNA concentration of the bacterium Ayurobst νad ^ ηае, [DNA bac] - the concentration of DNA of bacteria Lactobaculi 5 5 pp., [DNA of You] - the concentration of DNA of all bacteria;

calculated coefficients of ratios of bacterial concentrations: KC1 = 1d [DNA bac] - 1d ([DNA Ον] + [DNA Αν]),

KC2 = 1d [DNA of you] - 1d [DNA of bac],

KC3 = 1d [DNA of you] - 1d ([DNA Ον] + [DNA Αν]).

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table 2

The results obtained during the examination of 274 patients according to Amsel’s criteria, Nugent scores and real-time multiplex PCR

No. Amsel New gent 1DNAsu | 1DNA Αν) [DICK bae | | WPC you) KC1 KC2 KSZ one BV BV Ι, Ε + 08 8, Ε + 07 2, Ε + 05 3.Ε + 08 -2.89 3.16 0.28 2 BV BV 5.E + 06 Ι, Ε + 09 8, Ε + 07 7, Ε + 08 -1.17 0.92 -0.26 3 BV BV 5, E + O7 2, E7 3, Ε + 06 7.Ε + 07 -1.45 1.41 -0.04 4 BV BV 2.E + 08 oh yeah 6.Ε + 02 2.Ε + 07 -5.57 4,53 -1.04 5 BV BV 2.E + 08 Ι, Ε + 08 3, Ε + 05 3.Ε + 08 -2.96 3.06 0.10 6 BV BV 3, E + 07 Ι, Ε + 08 2, Ε + 06 9.Ε + 08 -1.85 2.59 0.74 7 BV BV 2.E + 09 2.EU8 Ι, Ε + 06 6.Ε + 08 -3.15 2.59 -0.56 8 BV BV 5, E + 07 2, ΕΗ) 7 2.Ε + 05 2, Ε + 08 -2.58 3.06 0.49 nine BV BV Z.E + 08 6, ΕΚ> 7 7, Ε + 05 Ι, Ε + 08 -2.79 2.24 -0.55 10 BV BV 2.E + 08 ό, ΕΗ) 6 1, Ε + 04 3.Ε + 07 -4.08 3.29 -0.79 eleven BV BV 5.E + 08 0, Ε + 00 3.Ε + 06 ό, Ε + 07 -2.22 1.25 -0.97 12 BV Fri 0, E + 00 Ο, Ε + ΟΟ 4, Ε + 07 3, Ε + Ο7 7.57 -0.11 7.46 thirteen BV BV 8, E + 04 7, EH5 2, Ε + 03 2, Ε + 06 -2.55 3.00 0.44 14 BV BV 6.E + 08 7, Ε ί-07 Ι, Ε + 05 9.Ε + 07 -3.70 2.81 -0.89 fifteen BV PT Ο, Ε + ΟΟ Ο, ΕΚΜ) Ι, Ε + 07 Ι, Ε + 07 7.16 -0.17 6.98 sixteen BV Fri Ο, Ε + ΟΟ 0.JEW0 3, Ε + 07 2, Ε + 07 7.52 -0.30 7.22 17 BV Fri 0, E + 00 Ο, ΕτΟΟ 1, Ε + 08 7.Ε + 07 8.08 -0.25 7.82 eighteen BV BV ό, Ε + 08 3, Ε4) 8 3, Ε + 06 9.B + 08 -2.47 2.44 -0.03 nineteen BV PT Ο, Ε + ΟΟ ο, Εωο Ι, Ε + 05 1, Ε + 0ό 5.11 1,03 6.14 twenty BV BV 8, E + 07 1.HE7 4, Ε + 05 Ι, Ε + 08 -2.37 2.43 0.06 21 BV PT Ο, Ε + ΟΟ Ο, ΕΗ, Ο 4, Ε + 08 2, Ε + 08 8.59 -0.21 8.38 22 BV BV Z.E + 05 Ο, Ε FROM 2, Ε + 05 2, Ε + 06 -0.06 0.94 0.88 23 BV PT 7, E + 06 4, Ε Yu5 4, Ε + 04 1, Ε + 06 -2.27 1.44 -0.83 24 BV BV 2.E + 08 1.HE7 ΰ, Ε + 04 3, Ε + 07 -3.52 2.68 -0.85 25 BV BV 1, E + 08 Ο, Ε Yu0 4, Ε + 06 2, Ε + 08 -1.50 0.19 1,69 26 BV BV Z.E + 08 7, Ε + 07 2.Ε + 06 3.Ε + 08 -2.26 2.17 -0.09 27 BV BV 5, E + 08 Ο, Ε 4) 7 8, Ε + 07 2.Ε + Ο8 -0.85 0.40 -0.46 28 OBV Fri 1, E + 03 Ο, Ει-ΟΟ 1, Ε + 09 5.Ε + 08 5.95 -0.32 5.63 29th BV Fri Ο, Ε + ΟΟ Ο, Ε + ΟΟ 1, Ε + 05 1, Ε + 06 5.08 0.96 6.03 thirty BV BV 1, E + 07 6, ΕηΟ7 7, Ε + Ο5 2.Ε + 08 -2.06 2,35 0.29 31 BV PT 4, E + 05 Ο, Ε Η00 Ι, Ε + 08 7.Ε + 07 2.41 -0.23 2.18 32 BV BV Ι, Ε + 07 4.HE7 ό, Ε + 05 3.Ε + 07 -1.94 1.73 -0.21 33 BV BV 1, E + 06 5, Ε Yu6 2, Ε + Ο5 2, Ε + 06 -1.42 0.84 -0.58 34 BV PT 0, E + 00 Ο, Ε 4) 0 3, Ε + 06 9.Ε + 05 6.52 -0.57 5.95 35 BV BV Ι, Ε + 08 9.HE6 ό, Ε + 05 4.Ε + 07 -2.27 1.85 -0.43 36 BV BV 3, Ε + Ο7 4, Ε 1-07 Ι, Ε + 07 5, Ε + 03 -0.81 1,62 0.81

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37 EV BV 9, E + 08 6, E + 07 8, Ε + 06 2, Ε + 08 -2.07 1.34 -0.74 38 BV pg Ο, Ε + 00 0, E + 00 2.Ε + 07 Ι, Ε + 07 7.33 -0.16 7.17 39 BV BV 3, E + 08 4, E + 07 3.Ε + 05 2.Ε + Ο8 -3.08 2.90 -0.18 40 BV BV 2, E + 08 Ι, Ε + 08 3.Ε + 07 Ι, Ε + 08 -1.03 0.52 -0.51 41 BV Fri 0, E + 00 0, E + 00 8.Ε + 06 Ι, Ε + 07 6.91 0.12 7.03 42 BV BV 4.E + 07 0, Ε + 00 2.Ε + 06 Ι, Ε + 08 -1.34 1.77 0.44 43 BV PT 0, E + 00 0, Ε + 00 3, Ε + 08 Ι, Ε + 08 8.40 -0.30 8.10 44 BV BV 2, E + 08 0, Ε + 00 2, Ε + 07 Ι, Ε + 08 -0.96 0.67 -0.29 45 BV BV 5, E + 06 Ι, Ε + 06 3.Ε + 03 5.Ε + 06 -3.37 3.26 -0.11 46 BV Fri 0, E + 00 0, Ε + 00 Ι, Ε + 08 5, Ε + 07 7.98 -0.30 7.68 47 BV BV 3, E + 06 6, Ε + 04 8, Ε + 06 5, Ε + 06 0.42 -0.16 0.26 48 BV BV 0, E + 00 0, Ε + 00 4, Ε + Ο8 2, Ε + 08 8.56 -0.32 8.24 49 BV BV Ι, Ε + 06 Ο, Ε + 00 1, Ε + 02 3, Ε + 06 -3.83 0.40 4.23 fifty OBV Fri Ο, Ε + 00 0, Ε + 00 Ι, Ε + 08 6, Ε + 07 8.10 -0.31 7.79 51 BV BV 8, E + 07 Ι, Ε + 07 5, Ε + 04 5, Ε + 07 -3.29 3.00 -0.30 52 BV BV Ι, Ε + 06 2, Ε + 06 Ι, Ε + 06 7.Ε + 06 -0.29 0.73 0.44 53 BV BV 3, E + 07 2, Ε + 06 Ι, Ε + 05 2, Ε + 07 -2.48 2.27 -0.21 54 BV BV 2.E + 06 2, Ε + 08 2, Ε + 06 3.Ε + 08 -2.00 2D5 0.15 55 BV BV Ι, Ε + 08 4, Ε + 07 3, Ε + 05 9, Ε + 07 -2.68 2.47 -0.21 56 BV BV 4.E + 08 6, Ε + 03 Ι, Ε + 05 2, Ε + 08 -3.42 3.06 -0.37 57 BV BV 5.E + 08 Ι, Ε + 08 4, Ε + 05 4, Ε + 08 -3.21 2.96 -0.26 58 BV BV 2, E + 09 9, Ε + 07 5, Ε + 07 8.Ε + 08 -1.67 1.26 -0.41 59 BV BV 4, E + 09 Ι, Ε + 08 4, Ε + 07 Ι, Ε + 09 -2.03 1,56 -0.47 60 BV BV Z.E + 09 2.Ε + 08 4, Ε + 07 Ι, Ε + 09 -1.87 1.39 -0.48 61 OBV OBV 0, E + 00 0, Ε + 00 Ι, Ε + 07 6, Ε + 06 7.10 -0.36 6.75 62 OBV OBV Ο, Ε + 00 0, Ε + 00 3, Ε + 08 2.Ε + 08 8.51 -0.21 8.29 63 OBV OBV 0.E + 00 0, Ε + 00 2.Ε + 06 8, Ε + 05 6.23 -0.32 5.91 64 OBV Fri 2, E + 08 0, Ε + 00 2, Ε + 03 2, Ε + 07 -5.07 4.08 -0.99 65 OBV Fri 0.E + 00 0, Ε + 00 3.Ε + 06 3.Ε + 07 6.50 0.90 7.40 66 OBV Fri 6.E + 04 0, Ε + 00 9.Ε + 06 7, Ε + 06 2.17 -0.08 2.09 67 OBV Fri Ο, Ε + 00 0, Ε + 00 4, Ε + 03 Ι, Ε + 06 3.65 2.40 6.05 68 OBV OBV Ο, Ε + 00 0, Ε + 00 8.Ε + 07 ό, Ε + 07 7.92 -0.12 7.79 69 OBV PT 0, E + 00 Ο, Ε + ΟΟ 4.Ε + 05 2, Ε + 06 5.56 0.81 6.37 70 OBV OBV 2, E + 03 Ο, Ε + 00 3, Ε + 08 2, Ε + 08 5.10 -0.10 5.00 71 OBV OBV Ο, Ε + 00 0, Ε + 00 5, Ε + 08 Z.E + 08 8.66 -0.19 8.47 72 OBV OBV Ι, Ε + 05 Ο, Ε + 00 Ι, Ε + 08 9.Ε + 07 3.15 -0.18 2.97 73 OBV OBV 1, E + 06 Ο, Ε + 00 3, Ε + 06 Ι, Ε + 07 0.38 0.75 1.13 74 OBV OBV 0, E + 00 Ο, Ε + 00 4, Ε + 08 3, Ε + 08 8.63 -0.15 8.49 75 OBV OBV 0, E + 00 Ο, Ε + ΟΟ 2, Ε + 07 Ι, Ε + 07 7.32 -0.19 7.13 76 OBV PT 0, E + 00 Ο, Ε + ΟΟ ό, Ε + 07 4, Ε + 07 7.77 -0.15 7.62 77 OBV OBV 0, E + 00 Ο, Ε + ΟΟ ό, Ε + 07 4, Ε + 07 7.77 -0.15 7.62 78 OBV PT 1, E + 08 3, Ε + 05 7.Ε + 06 9, Ε + 07 -1.26 1.09 -0.18 79 OBV OBV 2, E + 04 Ο, Ε +00 5.Ε + 06 3, Ε + 06 2.44 -0.25 2.19 80 OBV PT 0, E + 00 Ο, Ε + ΟΟ Ι, Ε + 06 3, Ε + 07 6.08 1.44 7.51 81 OBV PT 2, E + 03 4, Ε + 04 7, Ε + 04 2, Ε + 06 0.15 1.37 1,52 82 OBV OBV ό, Ε + 05 Ο, Ε +00 7, Ε + 06 5, Ε + 06 1.06 -0.15 0.91

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83 BV BV |, E + 08 2.Ε + 06 8, Ε + 04 4, Ε + 08 -3.14 0.59 3.73 84 Bb BV 3, E + 08 Ι, Ε + 07 Ι, Ε-ι 05 7, Ε + 07 -3.44 2.83 -0.62 85 OBV OBV 1, E + 05 2.Ε + 03 2, Ε + 08 2.Ε + 08 3.24 -0.16 3.09 86 OBV OBV Z.E + 06 4, Ε 4-03 1, Ε + 08 Ι, Ε + 08 1,69 -0.15 1,54 87 OBV PT 2, b + 09 2.Ε + 08 3, Ε + 07 4.Ε + 08 -1.96 1.13 -0.83 88 OBV OBV 0, E + 00 0, Ε + 00 Ι, Ε + 07 7.Ε + 06 7.13 -0.27 6.86 89 OBV Fri 1, E + 08 2.Ε + 08 2.Ε + 07 ό, Ε + 07 -1.26 0.57 -0.69 90 OBV OBV 2, E + 04 Ι, Ε + 03 ΐ, Ε + 09 5.Ε + 08 4.64 -0.28 4.36 91 OBV PT 2, E + 03 0, Ε + 00 1, Ε + 05 2.Ε + 08 1,62 3.20 4.81 92 OBV OBV 0, E + 00 0, Ε + 00 4, Ε + 07 2, Ε + 07 7.55 -0.28 7.27 93 OBV OBV 0, E + 00 Ο, Ε + 00 2, Ε + 08 2, Ε + 08 8.40 -0.21 8.19 94 BV BV 0, E + 00 Ι, Ε + 08 1, Ε + 05 4, Ε + 07 -2.91 2.49 -0.42 95 OBV OBV 4, E + 03 Ο, Ε + 00 Ι, Ε + 08 5, Ε + 07 4.44 -0.32 4.12 96 OBV OBV 0.E + 00 0, Ε + 00 2, Ε + 08 Ι, Ε + 08 8.31 -0.27 8.04 97 BV BV 2, E + 07 Ι, Ε + 06 5, Ε + 05 5, Ε + 06 -1.64 1,03 -0.61 98 OBV PT 3, E + 08 Ι, Ε + 07 5, Ε + 06 Ι, Ε + 08 -1.75 1.33 -0.42 99 OBV Fri Ι, Ε + 07 ό, Ε + 06 1, Ε + 04 ό, Ε + 06 -3.17 2.67 -0.50 one hundred OBV OBV 1, E + 05 9, Ε + 04 2, Ε + 07 Ι, Ε + 07 1.98 -0.28 1,69 101 OBV OBV 2.E + 07 0, Ε + 00 2, Ε + 08 8, Ε + 07 1.12 -0.40 0.72 102 OBV OBV 0, E + 00 Ο, Ε + 00 2, Ε + 06 8, Ε + 05 6.38 -0.46 5.92 103 OBV Fri 2, E + 07 5.Ε + 05 Ι, Ε + 08 4.Ε + 07 0.90 -0.47 0.42 104 OBV Fri 0, E + 00 2.Ε + 05 3, Ε + 08 2.Ε + 08 3.12 -0.27 2.85 105 OBV Fri 9, E + 07 Ο, Ε + 00 2.Ε + 08 9, Ε + 07 0.42 -0.41 0.01 106 OBV OBV 0, E + 00 Ο, Ε + 00 3, Ε + 08 Ι, Ε + 08 8.54 -0.51 8.03 107 BV BV 1, E + 09 9.Ε + 07 Ι, Ε + 07 5.Ε + 08 -2.05 1,67 -0.37 108 BV BV 5.E + 08 2.Ε + 07 2, Ε + 07 2.Ε + 08 -1.50 1,11 -0.39 109 OBV Fri Ο, Ε + 00 0, E0 Ι, Ε + 08 8, Ε + 07 8.12 -0.20 7.92 110 OBV OBV 0, E + (TO Ο, ΕΚΙΟ 2, Ε + 07 8, Ε + 06 7.35 -0.46 6.89 111 OBV OBV 0, E + 00 Ο, ΕιΌΟ 3.Ε + Ο8 Ι, Ε + 08 8.40 -0.32 8.08 112 OBV PT ό, Ε + 06 0, UEFA 5, Ε + 07 4, Ε + 07 0.91 -0.04 0.87 113 BV BV 6.Ε + 06 2, EY6 5 _τ Ε + 06 7, Ε + 06 -0.17 0.14 -0.03 114 BV BV Ι, Ε + 09 ό, Ε + 07 Ι, Ε + 07 7.Ε + 08 -1.94 1.68 -0.26 115 OBV OBV 5, Ε + 03 0, E0 5.Ε + 08 3, Ε + 08 4.95 -0.24 4.71 116 BV BV 2, Ε + 09 8.ΕΗ37 2, Ε + 07 7, Ε + 08 -1.93 1,56 -0.37 117 OBV OBV Ο, Ε + 00 Ο, Ε + 00 3.Ε + Ο5 Ι, Ε + 06 5.43 0.59 6.01 118 OBV PT 2.Ε + 05 Ο, ΕηΟΟ 2.Ε + 06 9, Ε + 0ό 1.06 0.64 1.70 119 BV BV 5, Ε + 0ό Ο, Ε + 00 Ι, Ε + 05 Ι, Ε + 07 -1.62 1.92 0.31 120 BV BV 5.Ε + 08 4, Ε1-07 2.Ε + 06 5, Ε + 07 -2.36 1.36 -1.00 121 BV BV Ι, Ε + 09 ό, Εΐ-07 Ι, Ε + 07 5, Ε + 08 -1.91 1,53 -0.39 122 OBV OBV 0, Ε + 00 0, Ε + 00 Ι, Ε + 07 5.Ε + 06 7.01 -0.28 6.73 123 OBV OBV 0, Ε + 00 Ο, Ε + 00 3.Ε + Ο5 Ι, Ε + 06 5.53 0.49 6.01 124 OBV OBV 0, Ε + 00 0, Ε + 00 Ι, Ε + 07 3, Ε + 06 7.01 -0.55 6.46 125 OBV OBV 0, Ε + 0Ο Ο, ΕηΟΟ Ι, Ε + 08 ό, Ε + 07 8.08 -0.32 7.76 126 OBV PT 0, Ε + 00 0, Ε + 00 7, Ε + 04 Ι, Ε + 06 4.82 1.19 6.01 127 OBV Fri 2, Ε + 03 Ο, ΕηΟΟ Ι, Ε + 03 Ι, Ε + 06 -0.20 2.97 2.77 128 OBV OBV 7.Ε + 03 0, Ε + 00 Ι, Ε + 07 5, Ε + 06 3.20 -0.35 2.85

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129 OBV PT ZX + 05 Z. EYU5 3, Ε + 05 Ι, Ε + 06 -0.19 0.46 0.27 130 OBV PT 0, E + 00 Ο, ΕΚΙΟ 3.Ε + 04 2, Ε + 06 4,54 1.80 6.33 131 OBV OBV 4, E + 05 Ο, Ε + 00 7.Γ + 08 4, Ε + 08 3.18 -0.20 2.98 132 OBV OBV 0, E + 00 Ο, Ε Κ0 2.Ε + 06 ό, Ε + 05 6.19 -0.40 5.78 133 OBV OBV 0, E + 00 Ο, Ε KO 1.Ε4 08 7, Ε + 07 8.15 -0.31 7.84 134 OBV OBV Oh, E + 0O Ο, Ε KO δ, Ε + 06 Ι, Ε + 07 6.93 0.23 7.15 135 OBV OBV 0, E + 00 Ο, Ε KO 3, Ε + 07 δ, Ε + 06 7.50 -0.60 6.90 136 OBV OBV 2, E + 04 Ο, Ε + 00 4, Ε + 06 2.Ε + 06 2.29 -0.21 2.09 137 OBV OBV 1, E + 06 5, ΕΚ) 3 3, Ε + 08 4.Ε + 08 2,55 0.04 2.59 138 OBV OBV Ο, Ε + 00 0. 2, Ε + 08 Ι, Ε + 08 8.21 -0.08 8.13 139 OBV OBV Ο, Ε + 00 Ο, Ε Κ0 7, Ε + 07 Ι, Ε + 07 7.84 -0.85 6.99 140 OBV OBV 0.E + 00 Ο, Ε + 00 Ι, Ε + 07 7, Ε + 06 7.02 -0.18 6.84 141 OBV OBV CE + 03 Ο, Ε Κ0 4, Ε + 08 2, Ε + 08 5.62 -0.30 5.32 142 OBV OBV Oh, E + 00 Ο, Ε + 00 8.Ε + 07 ό, Ε + 07 7.90 -0.09 7.81 143 OBV OBV 0.E + 00 Ο, ΕΚΙΟ 7.Ε + 07 7.Ε + 07 7.84 -0.03 7.81 144 OBV OBV 1. CLEAR Ο, Ε + 00 Ι, Ε + 08 Ι, Ε + 08 4.99 -0.06 4.93 145 OBV OBV 0, E + 00 Ο, ΕΚΙΟ 2.Ε + 07 Ι, Ε + 07 7.24 -0.24 7.00 146 OBV OBV 0.E + 00 Ο, ΕΚΙΟ 3.Ε + 07 2.Ε + Ο7 7.54 -0.14 7.39 147 OBV OBV Ο, Ε + 00 Ο, Ε Κ0 Ι, Ε + 07 7, Ε + 06 7.03 -0.18 6.84 148 OBV OBV 4, E + 05 Ο, ΕΚΙΟ 5, Ε + 06 3.Ε + 06 Ι, π -0.17 0.94 149 OBV OBV 0, E + 00 Ο, Ε Κ0 9, Ε + 07 7.Ε + 07 7.95 -0.08 7.87 150 OBV OBV 0, E KO Ο, ΕΚΙΟ 4, Ε + 07 3.Ε + 07 7.59 -0.07 7.51 151 OBV OBV Ο, Ε + 00 Ο, Ε Yu0 Ι, Ε + 08 8, Ε + 07 8.06 -0.14 7.92 152 OBV OBV 0, E + O0 Ο, Ε Щ0 Ι, Ε + 06 8, Ε + 05 6.08 -0.19 5.89 153 OBV OBV Ο, Ε + 00 Ο, Ε KO 8, Ε + 07 7, Ε + 07 7.90 -0.04 7.86 154 OBV OBV 0, E + 00 Ο, Ε KO 2, Ε + 06 2, Ε + 06 6.37 -0.17 6.19 155 OBV OBV 2, E + 03 Ο, Ε Yu0 Ι, Ε + 07 8, Ε + 06 3.77 -0.10 3.67 156 OBV OBV CE + 05 Ο, Ε IO 4.Ε + 07 3.Ε + 07 2.47 -0.15 2,32 157 OBV OBV 0.E + 00 Ο, ΕΚΙΟ 2, Ε + 07 Ι, Ε + 07 7.21 -0.18 7.03 158 OBV OBV 0.E + 00 0, UEFA 3.Ε + 08 2.Ε + 08 8.43 -0.06 8.37 159 OBV OBV 4> E + 04 0, E0 3.Ε + 08 3.Ε + 08 3.86 0,03 3.89 160 OBV OBV 4.E + 06 Ο, ΕΚΙΟ 8, Ε + 07 9.Ε + 07 1.26 0.05 1.31 161 OBV OBV 4, E + 03 1, ESL 8, Ε + 07 4, Ε + 07 4.23 -0.30 3.93 162 OBV OBV 6, E + O3 Ο, Ε KO 7, Ε + 08 Ι, Ε + 09 5.10 0.13 5.23 163 OBV OBV 0.E + 00 Ο, Ε KO 3.Ε + Ο6 2, Ε + 06 6.46 -0.22 6.24 164 OBV OBV 0, E + 00 Ο, Ε US 2, Ε + Ο5 Ι, Ε + 06 5.24 0.91 6.14 165 OBV OBV 6, E + 04 Ο, ΕηΟΟ 2, Ε + 08 2, Ε + 08 3.53 -0.09 3.44 166 OBV OBV Ο, Ε + 00 Ο, Ε KO 3, Ε + 08 2, Ε + 08 8.50 -0.27 8.23 167 OBV OBV 0, Ε + 0ΰ Ο, Ε KO 3, Ε + 08 4, Ε + 08 8.54 0.01 8.55 168 OBV OBV 0, E + 00 ο, ε then 1, Ε + 08 9, Ε + 07 8.06 -0.12 7.93 169 OBV OBV Ο, Ε + 00 ο, ε then Ι, Ε + 07 Ι, Ε + 07 7.10 -0.10 7.00 170 OBV OBV Ο, Ε + 00 ο, ε then 7, Ε + 07 3, Ε + Ο7 7.83 -0.29 7.54 171 OBV OBV 2, Ε + 03 Ο, Ε KO 2, Ε + 08 ό, Ε + 07 4.88 -0.39 4.49 172 OBV OBV 0, Ε + 00 Ο, Ε KO 2, Ε + 07 Ι, Ε + 07 7.26 -0.28 6.98 173 OBV OBV Ι, Ε + 03 Ο, Ε KO 2, Ε + 08 9, Ε + 07 5.21 -0.26 4.95 174 OBV OBV 2.Ε + Ο5 Ο, Ε KO 3, Ε + 07 2, Ε + 07 2.1, -0.17 1.94

- 11 025042

175 OBV OBV Ο, Ε + 00 0.BY0 8.E + 07 5.E + 07 7.91 -0.20 7.71 176 OBV OBV Ο, Ε + 00 0, E0 2.E + 04 1; E + 06 4.29 1.74 6.03 177 OBV OBV Ο, Ε + 00 0, E + 00 Ι, Ε + 08 1, E + O8 8.02 -0.01 8.01 178 OBV OBV Ο, Ε + 00 O. EYO 2.E + 08 2.E + 08 8.29 0,07 8.37 179 OBV OBV 3, E + 03 0, E0 4.E + 08 2.E + 08 5.04 -0.28 4.76 180 OBV OBV Ο, Ε + 00 0, E0 6.E + 07 4.E + 07 7.75 -0.10 7.65 181 OBV OBV Ι, Ε + 04 0, E0 Ι, Ε + 07 5.E + 06 2.85 -0.32 2,53 182 OBV OBV 2, E + 04 0, E + 00 7.E + 07 5.E + 07 3,58 -0.13 3.45 183 OBV OBV 0, E + 00 O. EYO Z.E + 08 Ι, Ε + 08 8.50 -0.38 8.12 184 OBV OBV 0, E + 00 0, E0 2, E + 08 2.E + 08 8.28 0.01 8.29 185 OBV OBV Z.E + OZ 0.JEW0 Z.E + 07 Z.E + 07 3.93 0.00 3.94 186 OBV OBV 0.E + 00 O. GUO 5, E + 08 4.E + 08 8.73 -0.11 8.61 187 OBV OBV Z.E + 04 0, E0 Ι, Ε + 07 8, E + 06 2.65 -0.13 2,51 188 OBV OBV 2.E + 04 0, E0 9.E + 06 4.E + 06 2.60 -0.32 2.28 189 OBV OBV 0.E + 00 oh hey 2, E + 06 8.E + 05 6.22 -0.30 5.92 190 OBV OBV Z.E + 04 0.JEW0 1.E + 08 9.E + 07 3.49 -0.03 3.46 191 OBV OBV 0.E + 00 0, E + 00 8.E + 0Y 7.E + 06 6.92 -0.07 6.85 192 OBV OBV 0, E + 00 2, E + 05 6.E + 08 2.E + 08 3,51 -0.43 3.07 193 OBV OBV 0, E + 00 0.JEW0 Z.E + 08 2.E + 08 8.52 -0.30 8.22 194 OBV OBV Ο, Ε + 00 Ο, Ε + 00 6.E + 05 Ι, Ε + 06 5.80 0.35 6.14 195 OBV OBV Ο, Ε + 00 0.JEW0 Z.E + 06 2, E + 08 6.45 1.90 8.34 196 OBV OBV 5, E + 04 0, E0 9.E + 05 2.E + 06 1.24 0.36 1,60 197 OBV OBV 7.E + 03 Ο, Ε + ΟΟ 4, E + 08 2, E + 08 4.73 -0.28 4.45 198 OBV OBV 1, E + 03 0, ECU 9, E + 04 1, E + 06 1.86 1.14 3.00 199 OBV OBV 1.E + 03 0.JEW0 2.E + 08 Ι, Ε + 08 5.20 -0.32 4.87 200 OBV OBV Ο, Ε + ΟΟ 0, E0 5.E + 06 4.E + 06 6.70 -0.09 6.62 201 OBV OBV Ι, Ε + 05 2, E + 04 1, E + 07 ], E + 07 1.86 0.00 1.86 202 OBV OBV 0, E + 00 O. EYO Z.E + 08 2.E + 08 8.45 -0.10 8.35 203 OBV OBV Ο, Ε + 00 O. EYO 8.E + 07 7.E + 07 7.91 -0.06 7.85 204 OBV OBV Ο, Ε + 00 0, E + 0O Ι, Ε + 06 8, E + 05 6.01 -0.10 5.91 205 OBV OBV 0, Ε + 0ΰ 0.JEW0 Z.E + 04 Ι, Ε + 06 4.41 1,61 6.03 206 OBV OBV Ο, Ε + 00 0.E + 00 2.E + 02 2.E + 06 2,30 4.07 6.37 207 OBV OBV 4.E + 05 2.E + 03 2.E + 08 Ι, Ε + 08 2.68 -0.11 2,56 208 OBV OBV Ο, Ε + 00 1.E + 07 Z.E + 07 Z.E + 07 0.30 0,03 0.33 209 OBV OBV 0.E + 00 0, E + 00 2.E + 08 2.E + 08 8.28 -0.09 8.19 210 OBV OBV 9.E + 03 Ο, Ε + 00 5.E + 03 8.E + 06 -0.23 3.19 2.96 211 OBV OBV 5.E + OZ 1.E + 03 2, E + 06 Ι, Ε + 06 2.46 -0.20 2.27 212 OBV OBV Z.E + 05 Ο, Ε + 00 Ι, Ε + 07 9.E + 06 1.51 -0.10 1.41 213 BV BV Z.E + 08 1, E8 2.E + 05 2.E + 08 -3.21 2.88 -0.33 214 BV BV Ι, Ε + 06 O, E + O0 5, E + 02 2.E + 06 -3.34 3.60 0.26 215 BV BV 4.E + 08 Ι, Ε + 08 6.E + 06 6, E + 08 -1.94 1.95 0.00 216 OBV Fri Ο, Ε + 00 0.E + 00 Ι, Ε + 05 4.E + 07 4.98 2.61 7.59 217 OBV OBV 0.E + 00 0.E + 00 9.E + 03 2.E + 06 3.95 2,35 6.30 218 BV BV 1, E + 07 Ο, Ε + ΟΟ Ι, Ε + 07 1, E + 07 0.12 -0.03 0.09 219 OBV Fri 3, E + 06 0.E + 00 Z.E + 06 2.E + 06 -0.01 -0.10 -0.11 220 OBV OBV 0.E + 00 Ο, Ε + 00 9.E + 05 2.E + 06 5.97 0.34 6.30

- 12 025042

221 OBV OBV Ο, Ε + 00 0, E + 0O Ι, Ε + 05 6, Ε + 06 5.17 1,57 6.74 222 OBV OBV 0.E + 00 0, E0 Ι, Ε + 05 2, Ε + 06 5.02 1.28 6.30 223 BV BV 1.E + 09 0, E + OO 3, Ε + 08 8.Ε + 08 -0.73 0.48 -0.25 224 OBV OBV 3, E + 03 Ο, Ε + 00 1, ΕΡ) 6 3.Ε + 06 2.71 0.29 3.01 225 OBV PT 1, E + 08 0, E + OO 2, Ε + 07 5.Ε + 07 -0.94 0.46 -0.48 226 OBV PT 6.E + 05 0, E + 00 9, Ε + 07 Ι, Ε + 08 2.19 0.02 2.21 227 OBV OBV 2, E + 05 0, E0 5, Ε + 06 3, Ε + 06 1,50 -0.20 1.30 228 OBV OBV 0, E + 00 ϋ, Ε + 00 4, Ε + 08 4, Ε + 08 8.57 0.02 8.59 229 OBV PT 2, E + 03 6.E + 07 5, Ε + 07 7.Ε + 07 -0.10 0.15 0.05 230 OBV OBV 1, E + 04 0 _t E + 00 3, Ε + 08 3, Ε + 08 4.44 -0.10 4.34 231 OBV OBV Ο, Ε + 00 0.JEW0 4, Ε + 06 3.Ε + 06 6.57 -0.11 6.46 232 OBV OBV 0, E + 00 0, Ε + 00 8, Ε + 05 2.Ε + 07 5.88 1.38 7.27 233 BV BV Z.E + 09 8.Ε + 07 6, Ε + 06 4, Ε + 08 -2.68 1.84 -0.84 234 OBV OBV Oh, E + 00 0, E0 5, Ε + 01 2, Ε + 06 1,69 4.61 6.30 235 OBV OBV 4.E + 04 Ο, ΕγΟΟ Ι, Ε + 06 7.Ε + 05 1.55 -0.24 1.31 236 BV BV 5, E + 09 0, E + 00 6, Ε + 04 7, Ε + Ο8 -4.91 4.10 -0.81 237 OBV OBV 4, E + 03 0, E + 00 9, Ε + 08 9, Ε + 08 5.31 0.00 5.32 238 OBV OBV 5, E + O3 Ο, Ε + 00 2, Ε + 08 2, Ε + 08 4,59 -0.04 4,55 239 OBV OBV 7, E + 03 Ο, Ε + 00 2, Ε + 07 Ι, Ε + 07 3.40 -0.21 3.19 240 OBV OBV b, E + 03 Ο, Ε + 00 6.Ε + 06 5, Ε + 06 3.02 -0.15 2.87 241 OBV OBV 5.E + 03 Ο, Ει-ΟΟ 7.Ε + 07 4.Ε + 07 4.20 -0.28 3.92 242 OBV OBV 7.E + 05 Ο, Ε + 00 Ι, Ε + 06 6, Ε + 05 0.16 -0.23 -0.07 243 OBV OBV 4, E + 03 Ο, ΕγΟΟ 4, Ε + 06 Ι, Ε + 07 3.01 0.42 3.43 244 OBV OBV Ι, Ε + 03 Ο, Ε + 00 3, Ε + 07 3, Ε + 07 4.42 -0.04 4.38 245 OBV OBV Ι, Ε + 03 Ο, ΕγΟΟ 3, Ε + Ο8 4, Ε + 08 5.50 0.09 5.59 246 OBV PT 8, E + 07 0, Ε + 00 3.Ε + 06 1, Ε + 07 -1.43 0.58 -0.85 247 OBV OBV 0.E + 00 Ο, Ε + 00 2, Ε + 04 1, Ε + 06 4.37 1.65 6.02 245 OBV Fri 2.E + 06 2, Ε * -04 Ι, Ε + 07 8, Ε + 0ό 0.82 -0.12 0.70 249 OBV OBV 3, E + 04 Ο, Ε + 00 5, Ε + 04 1, Ε + 06 0.27 1.32 1,59 250 OBV PT 6.E + 06 6.HE5 3.Ε + 06 5, Ε + 06 -0.38 0.25 -0.13 251 OBV Fri Ι, Ε + 07 |, ЕУ7 5, Ε + 06 2.Ε + 07 -0.69 0.62 -0.07 252 OBV OBV 2.E + 04 Ο, ΕγΟΟ Ι, Ε + 07 2, Ε + 07 2.74 0.32 3.06 253 OBV OBV 0, E + 00 Ο, Ε + 00 Ι, Ε + 07 2, Ε + 07 7.10 0.15 7.24 254 OBV OBV 5.E + 03 0, Ε + 00 Ι, Ε + 07 3, Ε + 07 3.43 0.35 3.78 255 OBV Fri 7, E + 07 0, E0 5, Ε + 07 9, Ε + 07 -0.15 0.26 0.11 256 OBV OBV Ο, Ε + 00 0, Ε + 00 4, Ε + 04 Ι, Ε + 06 4.60 1.42 6.02 257 OBV OBV Ι, Ε + 03 0, Ε + 00 β, Ε + 05 Ι, Ε + 06 2.83 0.10 2.94 258 OBV OBV 5, E + 03 Ο, Ε + 00 3.Ε + 08 3, Ε + 08 4.81 0.00 4.81 259 OBV OBV Oh, E + 0O 0, Ε + 00 3.Ε + 08 4, Ε + 08 8.49 0.11 8.60 260 OBV OBV 0, E + 00 0, Ε + 00 7.Ε + 05 Ι, Ε + 06 5.87 0.16 6.02 261 OBV OBV 0.E + 00 Ο, Ε + 00 2.Ε + 05 Ι, Ε + 06 5.40 0.63 6.02 262 OBV OBV 2.E + 03 Ο, Ε + 00 7, Ε + 06 Ι, Ε + 08 3.60 1.21 4.81 263 OBV OBV 0, E + 00 Ο, Ε + 00 2.Ε + 06 2, Ε + 06 6.36 -0.17 6.19 264 OBV OBV 0, E + 0O Ο, Ε + 00 4.Ε + 05 Ι, Ε + 06 5.57 0.45 6.02 265 OBV OBV 4, E + 04 Ο, Ε + 00 6.Ε + 05 Ι, Ε + 06 1.19 0.25 1.44 266 OBV PT Ο, Ε + 00 0, Ε + 00 Ι, Ε + 08 9, Ε + 07 7.98 -0.03 7.95 267 OBV OBV 1, E + 03 Ο, Ε + 00 8, Ε + 05 Ι, Ε + 06 2.90 0.09 2.99 268 OBV OBV 7, EGOZ Ο, ΕΓΟΟ Ι, Ε + 07 7, Ε + 06 3.19 -0.24 2.95 269 OBV OBV 4, E + 08 7, Ε + 07 4.Ε + 05 5.Ε + 08 -3.00 3.07 0,07 270 OBV OBV 2, E + 04 Ι, Ε + 03 Ι, Ε + 08 Ι, Ε + 08 3.74 -0.05 3.69 271 BV BV b, E + 07 3.Ε + 06 3, Ε + 04 3.Ε + 07 -3.34 3.08 -0.26 272 BV BV Z.E + 07 Ι, Ε + 07 3, Ε + 06 2.Ε + 07 -1.12 0.78 -0.34 273 BV BV 6, E + 03 4, Ε + 07 4, Ε + 0ό 2, Ε + 08 -0.99 1.76 0.78 274 OBV Fri 1.E + 04 2.Ε + 04 6.Ε + 04 Ι, Ε + 06 0.26 1.35 1,61

Further, for analysis, only those samples were selected for which matching results were obtained according to the Amsel criteria and Nugent scores (BV, N = 66, OBV, N = 156), Table. 3 and 4, respectively.

- 13 025042

Table 3

Real-time multiplex PCR test results for samples from 66 patients with bacterial vaginosis according to Amsel criteria and Nugent scores

No. Amsel New gent | DNASu | | DNA At | 1DNA bac [ 1DNA of you, KC1 KC2 KSZ one EV BV 1, E + 08 8.E + 07 2.Ε + 05 Z.E + 08 -2.89 3.16 0.28 2 BV BV 5, E + 06 Ι, Ε + 09 8.Ε + 07 7.Ε + 08 -1.17 0.92 -0.26 3 BV BV 5, E + 07 2.E + 07 3.Ε + 06 7.Ε + 07 -1.45 1.41 -0.04 4 BV BV 2.E + 08 0, E + 00 6, Ε + 02 2.Ε + 07 -5.57 4,53 -1.04 5 BV BV 2, E + 08 Ι, Ε + 08 3, Ε + 05 Z.E + 08 -2.96 3.06 0.10 6 BV BV Z.E + 07 Ι, Ε + 08 2, Ε + 06 9.Ε + 08 -1.85 2.59 0.74 7 BV BV 2, E + 09 2.Ε + 08 1, Ε + Ο6 ό, Ε + 08 -3.15 2.59 -0.56 8 BV BV 5.E + 07 2.Ε + 07 2.Ε + 05 2.Ε + 08 -2.58 3.06 0.49 nine BV BV Z.E + 08 6.HE7 7, Ε + 05 Ι, Ε + 08 -2.79 2.24 -0.55 10 BV BV 2.E + O8 β, Ε + 06 Ι, Ε + 04 3.Ε + 07 -4.08 3.29 -0.79 eleven BV BV 5.E + 08 Ο, Ε + ΟΟ 3.Ε + 06 6.Ε + 07 -2.22 1.25 -0.97 thirteen BV BV 8.E + 04 7.Ε + 05 2.Ε + 03 2.Ε + 06 -2.55 3.00 0.44 14 BV BV 6, E + 08 7, Ε + 07 Ι, Ε + 05 9, Ε + 07 -3.70 2.81 -0.89 eighteen BV BV 6, E + O8 3.Ε + Ο8 3.Ε + Ο6 9.Ε + 08 -2.47 2.44 -0.03 twenty BV BV 8.E + 07 Ι, Ε + 07 4.Ε + 05 Ι, Ε + 08 -2.37 2.43 0.06 22 BV BV 3, E + 05 0, Ε + 00 2.Ε + 05 2.Ε + 06 -0.06 0.94 0.88 24 BV BV 2, E + 08 Ι, Ε + 07 6.Ε + 04 3, Ε + 07 -3.52 2.68 -0.85 25 BV BV 1, E + 08 Ο, Ε + ΟΟ 4.Ε + 06 2, Ε + 08 -1.50 1,69 0.19 26 BV BV 3, E + 08 7.Ε + 07 2.Ε + 06 3.Ε + 08 -2.26 2.17 -0.09 27 BV BV 5, E + O8 ό, Ε + 07 8.Ε + 07 2.Ε + 08 -0.85 0.40 -0.46 thirty BV BV 1, E + 07 6, Ε + 07 7.Ε + 05 2.Ε + 08 -2.06 2,35 0.29 32 BV BV Ι, Ε + 07 4, Ε + 07 6.Ε + 05 3, Ε + Ο7 -1.94 1.73 -0.21 33 BV BV Ι, Ε + 06 5, Ε + 06 2, Ε + Ο5 2.Ε + 06 -1.42 0.84 -0.58 35 BV BV Ι, Ε + 08 9, Ε + 06 ό, Ε + 05 4, Ε + 07 -2.27 1.85 -0.43 36 BV BV 3, E + 07 4, Ε + 07 ί, Ε + 07 5.Ε + 08 -0.81 1,62 0.81

- 14 025042

37 BV BV 9, E + 08 6, E7 8, Εά 06 2, E + 08 -2.07 1.34 -0.74 39 BV BV Z.Y-08 4.E + 07 Z.E + 05 2, E + 08 -3.08 2.90 -0.18 40 BV BV 2, E4 08 1, E8 Z.E + 07 1, E + 08 -1.03 0.52 -0.51 42 BV Bb 4.HE7 0.E + 00 2.E + 06 Ι, Ε + 08 -1.34 1.77 0.44 44 BV BV 2, E + 08 0, E0 2.E + 07 Ι, Ε + 08 -0.96 0.67 -0.29 45 BV BV 5.E + 06 1, EY6 Z.E + OZ 5, E + 06 -3.37 3.26 -0.11 47 BV BV 3, E + 06 6, E4 8, E + 06 5.E + 06 0.42 -0.16 0.26 48 BV BV 0, E + 00 0, E0 4, E + 08 2, E + 08 8.56 -0.32 8.24 49 BV BV 1, E + 06 0, E0 1.E + 02 Z.E + 06 -3.83 4.23 0.40 51 BV BV 8, E + 07 1, E7 5, E + 04 5, E + 07 -3.29 3.00 -0.30 52 BV BV 1, E + 06 2, EY6 1, E + 06 7.E + 06 -0.29 0.73 0.44 53 BV BV Z.E + 07 2, EY6 1, E + 05 2, E + 07 -2.48 2.27 -0.21 54 BV BV 2, E + 0b 2, EH) 8 2.E + 06 Z.E + 08 -2.00 2.15 0.15 55 BV BV Ι, Ε + 08 4, E7 3, E + O5 9.E + 07 -2.68 2.47 -0.21 56 BV BV 4, E + 08 b.YEUZ 1, E + 05 2, E + 08 -3.42 3.06 -0.37 57 BV BV 5, E + 08 1, E8 4, E + 05 4, E + 08 -3.21 2.96 -0.26 58 BV BV 2.E + 09 9, E7 5.E + 07 8, E + 08 -1.67 1.26 -0.41 59 BV BV 4.E + 09 1.HE8 4, E + 07 Ι, Ε + 09 -2.03 1,56 -0.47 60 BV BV Z.E + 09 2, E + 08 4, E + 07 1, E + 09 -1.87 1.39 -0.48 83 BV BV Ι, Ε + 08 2, ES6 8.E + 04 4.E + 08 -3.14 3.73 0.59 84 BV BV Z.E + 08 1, E7 1, E + 05 7, E + 07 -3.44 2.83 -0.62 94 BV BV 0.E + 00 1, E8 1, E + 05 4, E + 07 -2.91 2.49 -0.42 97 BV BV 2, E + 07 1, EY6 5, E + 05 5.E + 06 -1.64 1,03 -0.61 107 BV BV Ι, Ε + 09 9.HE7 1.E + 07 5, E + 08 -2.05 1,67 -0.37 108 BV BV 5, E + 08 2, E7 2.E + 07 2, E + 08 -1.50 1,11 -0.39 113 BV BV 6, E + O6 2, EY6 5.E + 06 7, E + 06 -0.17 0.14 -0.03 114 BV BV Ι, Ε + 09 6, E7 1.E + 07 7, E + 08 -1.94 1.68 -0.26 116 BV BV 2.E + 09 8.HE7 2.E + 07 7 _? E + 08 -1.93 1,56 -0.37 119 BV BV 5, E + 06 0, E0 1.E + 05 Ι, Ε + 07 -1.62 1.92 0.31 120 BV BV 5, E + 08 4, E1-07 2.E + 06 5, E + 07 -2.36 1.36 -1.00 121 BV BV 1, E + 09 6, E U7 1, E + 07 5, E + 08 -1.91 1,53 -0.39 213 BV BV 3, E + 08 1.HE8 2, E + 05 2, E + 08 -3.21 2.88 -0.33 214 BV BV 1, E + 06 0, E SO 5, E + 02 2, E + 06 -3.34 3.60 0.26 215 BV BV 4, E + 08 1.EN08 6, E + 06 6, E + 08 -1.94 1.95 0.00 218 BV EV 1, E + 07 0 _; EOOOO Ι, Ε + 07 Ι, Ε + 07 0.12 -0.03 0.09 223 BV BV 1, E + 09 0, E + 00 Z.E + 08 8, E + 08 -0.73 0.48 -0.25 233 BV BV 3, E + 09 8.E + 07 6, E + 06 4, E + 08 -2.68 1.84 -0.84 236 BV BV 5, E + 09 0.E1-00 6, E + 04 7, E + 08 -4.91 4.10 -0.81 271 BV BV 6.E + 07 3, EY6 3, E + 04 3, E + 07 -3.34 3.08 -0.26 272 BV BV Z.E + 07 1.HE7 3, E + 06 2.E + 07 -1.12 0.78 -0.34 273 BV BV 6.E + 03 4.E U7 4, E + 06 2, E + 08 -0.99 1.76 0.78

- 15 025042

Table 4

Results of real-time multiplex PCR testing of samples from 156 patients without bacterial vaginosis according to Amsel criteria and Nugent scores

No. Amsel Nyod- lamb | DNASu | ! ΑΙ4ΚΑν | | DNA Bass | [DNA of you] KC1 KC2 ksz 61 OBV OBV 0, E + 00 0, Ε + 00 Ι, Ε + 07 6.Ε + 06 7.10 -0.36 6.75 62 OBV OBV 0, E + 00 0, Ε + 00 3, Ε + 08 2.Ε + 08 8.51 -0.21 8.29 63 OBV OBV Ο, Ε + 00 0, Ε + 00 2, Ε + 06 8.Ε + 05 6.23 -0.32 5.91 68 OBV OBV Ο, Ε + 00 0, Ε + 00 8, Ε + 07 6, E + 07 7.92 -0.12 7.79 70 OBV OBV 2.E + OZ Ο, Ε + 00 3, Ε + 08 2.Y + 08 5.10 -0.10 5.00 71 OBV OBV 0.E + 00 0, Ε + 00 5.Ε + 08 Z.E + 08 8.66 -0.19 8.47 72 OBV OBV 1, E + 05 Ο, Ε + 00 Ι, Ε + 08 9, E + 07 3.15 -0.18 2.97 73 OBV OBV Ι, Ε + 06 0, Ε + 00 3.Ε + 06 Ι, Ε + 07 0.38 0.75 1.13 74 OBV OBV Ο, Ε + ϋΟ 0, Ε + 0Ο 4, Ε + 08 Z.E + 08 8.63 -0.15 8.49 75 OBV OBV 0, E + 00 0, Ε + 00 2.Ε + 07 Ι, Ε + 07 7.32 -0.19 7.13 77 OBV OBV 0, E + 00 0, Ε + 00 6, Ε + 07 4, E + 07 7.77 -0.15 7.62 79 BOTH OBV 2.Ε + 04 0, Ε + 00 5, Ε + 06 Z.E + 06 2.44 -0.25 2.19 82 OBV OBV 6, Ε + 05 0, Ε + 00 7, Ε + 06 5.E + 06 1.06 -0.15 0.91 85 OBV OBV Ι, Ε + 05 2.Ε + 03 2, Ε + 08 2, E + 08 3.24 -0.16 3.09 86 OBV OBV 3, Ε + 06 4, Ε + 03 Ι, Ε + 08 Ι, Ε + 08 1,69 -0.15 1,54 88 OBV OBV Ο, Ε + 00 0, Ε + 00 Ι, Ε + 07 7.E + 06 7.13 -0.27 6.86 90 OBV OBV 2.Ε + 04 Ι, Ε + 03 Ι, Ε + 09 5.E + 08 4.64 -0.28 4.36 92 OBV OBV 0, Ε + 00 0, Ε + 00 4, Ε + 07 2, E + 07 7.55 -0.28 7.27 93 OBV OBV Ο, Ε + Οΰ Ο, Ε + 00 2, Ε + 08 2, E + 08 8.40 -0.21 8.19 95 OBV OBV 4, Ε + 03 Ο, Ε + 00 Ι, Ε + 08 5, E + 07 4.44 -0.32 4.12 96 OBV OBV 0, Ε + 00 0, Ε + 00 2, Ε + 08 1, E + 08 8.31 -0.27 8.04 one hundred OBV OBV Ι, Ε + 05 9, Ε + 04 2, Ε + 07 3, E + 07 1.98 -0.28 1,69 101 OBV OBV 2, Ε + 07 0, Ε + 00 2, Ε + 08 8, E + 07 1.12 -0.40 0.72 102 OBV OBV Ο, Ε + 00 0, Ε + 00 2, Ε + 06 8.E + 05 6.38 -0.46 5.92 106 OBV OBV 0, Ε + 00 Ο, Ε + 00 3, Ε + Ο8 1, E + 08 8.54 -0.51 8.03 by OBV OBV 0, Ε + 00 0, Ε + 00 2.Ε + 07 8.E + 06 7.35 -0.46 6.89 111 OBV OBV 0, Ε + 00 Ο, Ε + 00 3, Ε + 08 Ι, Ε + 08 8.40 -0.32 8.08 115 OBV OBV 5, Ε + 03 0, Ε + 00 5.Ε + 08 Z.E + 08 4.95 -0.24 4.71 117 OBV OBV Ο, Ε + 00 ΰ, Ε + 00 3, Ε + Ο5 1, E + 06 5.43 0.59 6.01 122 OBV OBV 0, Ε + 00 Ο, Ε + 00 1 _τ Ε + 07 5, E + 06 7.01 -0.28 6.73 123 OBV OBV 0, Ε + 00 ΰ, Ε + 00 3.Ε + Ο5 Ι, Ε + 06 5.53 0.49 6.01 124 OBV OBV Ο, Ε + 00 ΰ, Ε + 00 Ι, Ε + 07 Z.E + 06 7.01 -0.55 6.46 125 OBV OBV Ο, Ε + 00 0, Ε + 00 Ι, Ε + 08 6, E + 07 8.08 -0.32 7.76 128 OBV OBV 7, Ε + 03 Ο, Ε + 00 Ι, Ε + 07 5.E + 06 3.20 -0.35 2.85 131 OBV OBV 4, Ε + 05 0, Ε + 00 7, E + 08 4, E + 08 3.18 -0.20 2.98 132 OBV OBV 0, Ε + 00 Ο, Ε + 00 2, Ε + 0ό ΰ, Ε + 05 6.19 -0.40 5.78 133 OEV OBV 0, Ε + 00 Ο, Ε + 00 Ι, Ε + 08 7, E + 07 8.15 -0.31 7.84 134 OBV OBV ΰ, Ε + 00 0, Ε + 0ΰ 8, Ε + 06 1, E + 07 6.93 0.23 7.15 135 OBV OBV 0, Ε + 00 0, Ε + 00 3.Ε + 07 8, E + 06 7.50 -0.60 6.90 136 OBV OBV 2, Ε + 04 Ο, Ε + 00 4.Ε + 06 2, E + O 2.29 -0.21 2.09

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137 OBV OBV Ι, Ε + 06 5.E + 03 3.Ε + 08 4, Ε + 08 2,55 0.04 2.59 138 OBV OBV 0 _? E + 00 0.E + 00 2, Ε + 08 Ι, Ε + 08 8.21 -0.08 8.13 139 OBV OBV 0, E0 Ο, Ε + 00 7, Ε + 07 Ι, Ε + 07 7.84 -0.85 6.99 140 OBV OBV O> E + 0O 0.E + 00 Ι, Ε + 07 7, Ε + 06 7.02 -0.18 6.84 141 OBV OBV 1, E + 03 0, E + 00 4, Ε + 08 2, Ε + 08 5.62 -0.30 5.32 142 OBV OBV Ο, Ε + 00 Ο, Ε + ΟΟ β, Ε + 07 6.Ε + 07 7.90 -0.09 7.81 143 OBV OBV 0, E + 00 0, E + 00 7.Ε + 07 7, Ε + 07 7.84 -0.03 7.81 144 OBV OBV 1 ₊ E + 03 0, E + 00 Ι, Ε + 08 Ι, Ε + 08 4.99 -0.06 4.93 145 OBV OBV Ο, Ε + 00 0, E + 00 2, Ε + 07 Ι, Ε + 07 7.24 -0.24 7.00 146 OBV OBV Ο, Ε + 00 0, E + 00 3, Ε + 07 2, Ε + 07 7.54 -0.14 7.39 147 OBV OBV 0, E + 00 0.E + 00 Ι, Ε + 07 7, Ε + 06 7.03 -0.18 6.84 148 OBV OBV 4.E + 05 0, E + 00 5, Ε + 06 3.Ε + 06 1,11 -0.17 0.94 149 OBV OBV 0.E + 00 0, E4 00 9.Ε + 07 7, Ε + 07 7.95 -0.08 7.87 150 OBV OBV 0.E + 00 Ο, Ε + 00 4.Ε + 07 3.Ε + 07 7.59 -0.07 7.51 151 OBV OBV Ο, Ε + 00 Ο, Ε + 00 Ι, Ε + 08 8, Ε + 07 8.06 -0.14 7.92 152 OBV OBV O.E + OO Ο, Ε + 00 Ι, Ε + 06 8, Ε + Ο5 6.08 -0.19 5.89 153 OBV OBV Ο, Ε + ΟΟ Ο, Ε + ΟΟ 8.Ε + 07 7.Ε + 07 7.90 -0.04 7.86 154 OBV OBV 0, E + 00 Ο, Ε + 00 2.Ε + 06 2, Ε + 06 6.37 -0.17 6.19 155 OBV OBV 2, E + 03 Ο, Ε + 00 Ι, Ε + 07 8, Ε + 06 3.77 -0.10 3.67 156 OBV OBV 1, E + 05 Ο, Ε + 00 4.Ε + 07 3, Ε + 07 2.47 -0.15 2,32 157 OBV OBV Ο, Ε + ΟΟ Ο, ΕτΟΟ 2.Ε + 07 Ι, Ε + 07 7.21 -0.18 7.03 158 OBV OBV Oh, E + 0O Ο, Ε + 00 3.Ε + 08 2, Ε + 08 8.43 -0.06 8.37 159 OBV OBV 4, E + 04 ο, ε + οο 3.Ε + 08 3, Ε + 08 3.86 0,03 3.89 160 OBV OBV 4, E + 0b Ο, Ε + ΟΟ Ε, Ε + 07 9, Ε + 07 1.26 0.05 1.31 161 OBV OBV 4.E + 03 1.Ε403 8, Ε + 07 4, Ε + 07 4.23 -0.30 3.93 162 OBV OBV b, E + 03 Ο, Ε + 00 7.Ε + 08 Ι, Ε + 09 5.10 0.13 5.23 163 OBV OBV 0, E + 00 0, Ε4 00 3.Ε + 06 2.Ε + 06 6.46 -0.22 6.24 164 OBV OBV 0, E + 00 Ο, Ε-ίΟΟ 2, Ε + 05 Ι, Ε + 06 5.24 0.91 6.14 165 OBV OBV b.E + 04 Ο, Ε + 00 2, Ε + 08 2, Ε + 08 3.53 -0.09 3.44 166 OBV OBV Ο, Ε + ΟΟ Ο, Ε + 00 3.Ε + 08 2.Ε + 08 8.50 -0.27 8.23 167 OBV OBV 0, E + 00 0, Ε40Ο 3.Ε + 08 4.Ε + 08 8.54 0.01 8.55 168 OBV OBV 0, E + 00 Ο, Ε + 00 Ι, Ε + 08 9, Ε + 07 8.06 -0.12 7.93 169 OBV OBV 0, E + 00 Ο, Ε + 00 Ι, Ε + 07 Ι, Ε + 07 7.10 -0.10 7.00 170 OBV OBV 0, E + 00 Ο, Ε + ΟΟ 7, Ε + 07 3, Ε + 07 7.83 -0.29 7.54 171 OBV OBV 2, E + 03 Ο, Ε + 00 2, Ε + 08 6, Ε + 07 4.88 -0.39 4.49 172 OBV OBV 0, E + 00 0, Ε + 00 2.Ε + 07 Ι, Ε + 07 7.26 -0.28 6.98 173 OBV OBV 1.E + 03 0, Ε + ΘΟ 2.Ε + 08 9.Ε + 07 5.21 -0.26 4.95 174 OBV OBV 2, E + 05 Ο, Ε + 00 3, Ε + 07 2.Ε + 07 2D1 -0.17 1.94 175 OBV OBV 0.E + 00 0, Ε + 00 8.Ε + 07 5, Ε + 07 7.91 -0.20 7.71 176 OBV OBV Ο, Ε + 00 0, Ε + 00 2.Ε + 04 Ι, Ε + 06 4.29 1.74 6.03 177 OBV OBV Ο, Ε + 00 0, Ε + 00 Ι, Ε + 08 Ι, Ε + 08 8.02 -0.01 8.01 178 OBV OBV 0, E + 00 Ο, Ε + ΟΟ 2.Ε + 08 2, Ε + 08 8.29 0,07 8.37 179 OBV OBV Z.E + OZ 0, Ε + 00 4, Ε + 08 2, Ε + 08 5.04 -0.28 4.76 180 OBV OBV 0, E + 00 Ο, Ε + 00 6, Ε + 07 4, Ε + 07 7.75 -0.10 7.65 181 OBV OBV 1, E + 04 0, Ε + 00 Ι, Ε + 07 5.Ε + 06 2.85 -0.32 2,53 182 OBV OBV 2, E + 04 Ο, Ε + 00 7.Ε + 07 5.Ε + 07 3,58 -0.13 3.45

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183 OBV OBV O, E + O0 0, E + 00 3.Ε + 08 Ι, Ε + 08 8.50 -0.38 8.12 184 OBV OBV 0, E + 00 Ο, Ε + 00 2, Ε + 08 2.Ε + 08 8.28 0.01 8.29 185 OBV OBV Z.E + OZ Ο, Ε + 00 3.Ε + 07 3, Ε + 07 3.93 0.00 3.94 186 OBV OBV 0, E + 00 Ο, Ε + 00 5, Ε + 08 4, Ε + 08 8.73 -0.11 8.61 187 OBV OBV 3, E + 04 Ο, Ε + 00 Ι, Ε + 07 8.Ε + 06 2.65 -0.13 2,51 188 OBV OBV 2, E + 04 Ο, Ε + ΟΟ 9, Ε + 06 4, Ε + ϋό 2.60 -0.32 2.28 189 OBV OBV 0, E + 00 Ο, Ε + ΟΟ 2.Ε + 06 8.Ε + 05 6.22 -0.30 5.92 190 OBV OBV 3, E + 04 Ο, Ε + 00 Ι, Ε + 08 9.Ε + 07 3.49 -0.03 3.46 191 OBV OBV 0, E + 00 ο, ε + οο δ, Ε + 06 7.Ε + 06 6.92 -0.07 6.85 192 OBV OBV 0, E + 00 2.Ε + 05 ό, Ε + 08 2, Ε + 08 3,51 -0.43 3.07 193 OBV OBV Ο, Ε + 00 Ο, Ε + 00 3, Ε + 08 2, Ε + 08 8.52 -0.30 8.22 194 OBV OBV Ο, Ε + 00 Ο, Ε + 00 ό, Ε + 05 Ι, Ε + 06 5.80 0.35 6.14 195 OBV OBV Ο, Ε + 00 Ο, Ε + ΟΟ 3.Ε + 06 2.Ε + 08 6.45 1.90 8.34 196 OBV OBV 5, E + 04 0, Ε + 00 9, Ε + 05 2.Ε + 06 1.24 0.36 1,60 197 OBV OBV 7, E + 03 Ο, Ε + 00 4, Ε + 08 2.Ε + 08 4.73 -0.28 4.45 198 OBV OBV Ι, Ε + 03 0 _} Ε + 00 9, Ε + 04 Ι, Ε + 06 1.86 1.14 3.00 199 OBV OBV 1, E + 03 Ο, Ε + 00 2.Ε + 08 Ι, Ε + 08 5.20 -0.32 4.87 200 OBV OBV 0, E + 00 Ο, Ε + 00 5.Ε + 06 4, Ε + 06 6.70 -0.09 6.62 201 OBV OBV Ι, Ε + 05 2, Ε + 04 Ι, Ε + 07 Ι, Ε + 07 1.86 0.00 1.86 202 OBV OBV Ο, Ε + 00 Ο, Ε + 00 3, Ε + 08 2, Ε + 08 8.45 -0.10 8.35 203 OBV OBV Ο, Ε + 00 Ο, Ε + ΟΟ 8, Ε + 07 7, Ε + 07 7.91 -0.06 7.85 204 OBV OBV 0, E + 00 Ο, Ε + 00 Ι, Ε + 06 8, Ε + 05 6.01 -0.10 5.91 205 OBV OBV 0, E + 00 Ο, Ε + 00 3.Ε + 04 Ι, Ε + 06 4.41 1,61 6.03 206 OBV OBV Ο, Ε + ΟΟ Ο, Ε + ΟΟ 2, Ε + 02 2, Ε + 06 2,30 4.07 6.37 207 OBV OBV 4.E + 05 2.Ε + 03 2.Ε + 08 Ι, Ε + 08 2.68 -0.11 2,56 208 OBV OBV Ο, Ε + 00 1, Ε + 07 3.Ε + 07 3, Ε + Ο7 0.30 0,03 0.33 209 OBV OBV 0, E + 00 Ο, Ε + 00 2, Ε + 08 2, Ε + 08 8.28 -0.09 8.19 210 OBV OBV 9.E + 03 Ο, Ε + ΟΟ 5, Ε + 03 8, Ε + 06 -0.23 3.19 2.96 211 OBV OBV 5.E + OZ Ι, Ε + 03 2.Ε + 06 Ι, Ε + 06 2.46 -0.20 2.27 212 OBV OBV Z.E + 05 Ο, Ε + 00 ! , Ε + 07 9.Ε + 06 1.51 -0.10 1.41 217 OBV OBV Ο, Ε + 00 Ο, Ε + 00 9, Ε + 03 2, Ε + 06 3.95 2,35 6.30 220 OBV OBV 0, E + 00 0, Ε + 00 9, Ε + 05 2, Ε + 06 5.97 0.34 6.30 221 OBV OBV 0, E + 00 Ο, Ε + 00 Ι, Ε + 05 6, Ε + 06 5.17 1,57 6.74 222 OBV OBV 0, E + 00 Ο, Ε + 00 Ι, Ε + 05 2, Ε + 06 5.02 1.28 6.30 224 OBV OBV 3, E + O3 Ο, Ε + 00 Ι, Ε + 06 3, Ε + 06 2.71 0.29 3.01 227 OBV OBV 2.E + 05 Ο, Ε + 00 5.Ε + 06 3, Ε + 06 1,50 -0.20 1.30 228 OBV OBV Ο, Ε + 00 Ο, Ε + 00 4.Ε + 08 4, Ε + 08 8.57 0.02 8.59 230 OBV OBV 1, E + 04 Ο, Ε + 00 3.Ε + Ο8 3, Ε + 08 4.44 -0.10 4.34 231 OBV OBV Ο, Ε + 00 Ο, Ε + 00 4.Ε + 06 3, Ε + 06 6.57 -0.11 6.46 232 OBV OBV Ο, Ε + 00 Ο, Ε + 00 8, Ε + 05 2, Ε + 07 5.88 1.38 7.27 234 OBV OBV Ο, Ε + ΟΟ Ο, Ε + ΟΟ 5, Ε + 01 2, Ε + 06 1,69 4.61 6.30 235 OBV OBV 4, E + 04 Ο, Ε + 00 Ι, Ε + 06 7, Ε + 05 1.55 -0.24 1.31 237 OBV OBV 4.E + 03 0, Ε + 00 9, Ε + 08 9, Ε + Ο8 5.31 0.00 5.32 238 OBV OBV 5.E + 03 Ο, Ε + 00 2.Ε + 08 2, Ε + 0 " 4,59 -0.04 4,55 239 OBV OBV 7.E + 03 Ο, Ε + 00 2.Ε + 07 Ι, Ε + 07 3.40 -0.21 3.19 240 OBV OBV 6.E + 03 Ο, Ε + 00 ό, Ε + 06 5, Ε + 06 3.02 -0.15 2.87

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241 OBV OBV 5.E + 03 Ο, Ε + ΟΟ 7, Ε + 07 4, Ε + 07 4.20 -0.28 3.92 242 OBV OBV 7.E + 05 Ο, Ε + ΟΟ Ι, Ε + 06 6.Ε + 05 0.16 -0.23 -0.07 243 OBV OBV 4.E + 03 Ο, Ε + ΟΟ 4.Ε + 06 Ι, Ε + 07 3.01 0.42 3.43 244 OBV OBV Ι, Ε + 03 Ο, Ε + ΟΟ 3.Ε + 07 3, Ε + 07 4.42 -0.04 4.38 245 OBV OBV Ι, Ε + 03 Ο, Ε + ΟΟ 3, Ε + 08 4, Ε + 08 5.50 0.09 5.59 247 OBV OBV Ο, Ε + ΟΟ Ο, Ε + ΟΟ 2, Ε + 04 Ι, Ε + 06 4.37 1.65 6.02 249 OBV OBV Z.E + 04 Ο, Ε + ΟΟ 5, Ε + 04 Ι, Ε + 06 0.27 1.32 1,59 252 OBV OBV 2.E + 04 Ο, Ε + ΟΟ 1, Ε + 07 2, Ε + 07 2.74 0.32 3.06 253 OBV OBV Ο, Ε + ΟΟ 0, Ε + 00 Ι, Ε + 07 2, Ε + 07 7.10 0.15 7.24 254 OBV OBV 5.E + 03 0, Ε + 00 1, Ε + 07 3, Ε + 07 3.43 0.35 3.78 256 OBV OBV Ο, Ε + ΟΟ Ο, Ε + ΟΟ 4, Ε + 04 Ι, Ε + 06 4.60 1.42 6.02 257 OBV OBV Ι, Ε + 03 Ο, Ε + ΟΟ 8, Ε + 05 Ι, Ε + 06 2.83 0.10 2.94 258 OBV OBV 5, Ε + 03 ΰ, Ε4 00 3, Ε + Ο8 3, Ε + 08 4.81 0.00 4.81 259 OBV OBV Ο, Ε + ΟΟ Ο, Ε + ΟΟ 3.Ε + 08 4.Ε + 08 8.49 0.11 8.60 260 OBV OBV Ο, Ε + ΟΟ Ο, Ε + ΟΟ 7.Ε + 05 Ι, Ε + 06 5.87 0.16 6.02 261 OBV OBV Ο, Ε + ΟΟ Ο, Ε + ΟΟ 2.Ε + 05 Ι, Ε + 06 5.40 0.63 6.02 262 OBV OBV 2.Ε + 03 Ο, Ε + ΟΟ 7.Ε + 06 Ι, Ε + 08 3.60 1.21 4.81 263 OBV OBV Ο, Ε + ΟΟ Ο, Ε + ΟΟ 2, Ε + 06 2.Ε + 06 6.36 -0.17 6.19 264 OBV OBV Ο, Ε + ΟΟ Ο, Ε + ΟΟ 4.Ε + 05 Ι, Ε + 06 5.57 0.45 6.02 265 OBV OBV 4.Ε + 04 Ο, Ε + ΟΟ 6.Ε + 05 Ι, Ε + 06 1.19 0.25 1.44 267 OBV OBV Ι, Ε + 03 Ο, Ε + ΟΟ 8, Ε + 05 Ι, Ε + 06 2.90 0.09 2.99 268 OBV OBV 7.Ε + 03 Ο, Ε + ΟΟ 1, Ε + 07 7.Ε + 06 3.19 -0.24 2.95 269 OBV OBV 4.Ε + 08 7, Ε + 07 4, Ε + 05 5.Ε + 08 -3.00 3.07 0,07 270 OBV OBV 2, Ε + 04 ΐ, Ε-SW 1, Ε + 08 1, Ε + 08 3.74 -0.05 3.69

From the table. 2-4 it is seen that for all samples the value of [DNA of you] exceeds 10 ⁵ GE / ml, i.e. it is not possible to interpret values below this threshold; therefore, [DNA of you] 10 ⁵ GE / ml was taken as the lower threshold of sample validity.

Further, the normal number of bacteria on the vaginal mucosa of a woman of reproductive age was determined. To do this, we evaluated the distribution of patients by the total number of bacteria and by cutting off five percent of the smallest values, the lower limit of normal [DNA of you] 10 ⁶ GE / ml was adopted (Fig. 1).

Thus, based on the above data, as well as statistical processing of the calculated ratios of the concentrations of bacteria, the criteria for differential diagnosis of the absence of bacterial vaginosis were formed, including a special case of the absence of bacterial vaginosis - a decrease in the degree of bacterial contamination of the vagina, the intermediate state of microflora and bacterial vaginosis.

At 1§ [DNA of You]> 5, KC1> 1 and KC2 <1, the absence of bacterial vaginosis is noted, while at 5 <1d [DNA of You] <6, an additional decrease in the degree of bacterial contamination of the vagina is noted.

At 1§ [DNA of You]> 5, 0.5 <KC1 <1, KC2 <1 and KC3 <2, an intermediate state of microflora is observed.

At 1§ [DNA of you]> 5, KC1 <0.5 and KC3 <2, bacterial vaginosis is diagnosed.

At 1§ [DNA of you] <5, it is noted that the number of bacteria is insufficient for analysis.

When stating the intermediate state of microflora, the patient is additionally determined to have complaints of pathological discharge and pH of the discharge. At pH <4.5 and the absence of complaints of pathological discharge, the patient is recommended to repeat the analysis in the next menstrual cycle, and at pH> 4.5 and the presence of complaints of pathological discharge, the patient is diagnosed with bacterial vaginosis.

The total data on the distribution of conclusions obtained using the extended gold standard and the method according to the invention are presented in table. 5.

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The results obtained using the extended gold standard and the method according to the invention

Table 5

Conclusion (“extended gold standard") Number of patients Conclusion (method but invention) Bacterial vaginosis 66 65 bacterial vaginosis 1 lack of bacterial vaginosis Lack of bacterial vaginosis 156 152 absence bacterial vaginosis 4 bacterial vaginosis

Thus, the sensitivity of the method according to the invention for the diagnosis of bacterial vaginosis was 98.5%, specificity 97.4%.

At the second stage of development of a method for differential diagnosis of imbalances in the vaginal microflora, samples were selected for further analysis, the decrease in the number of lactobacilli in which could not be explained by the presence of BV-associated microorganisms, N = 22 (Table 6).

Table 6

The results obtained in the examination of 22 patients who have a violation of the balance of the vaginal microflora, not explained by the presence of BV-associated microorganisms, using Amsel criteria, Nugent scores and real-time multiplex PCR method

No. Amsel New gent | DNASu] (DNA Αν] | DNA 1.ac | (DNA of you | KC1 KC2 KSZ nineteen BV Fri 0, E + 00 0, EIU Ι, Ε + 05 Ι, Ε + 06 5.11 1,03 6.14 67 OBV Fri 0, E + 00 0.E + 00 4.Ε + 03 1, Ε + Οό 3.65 2.40 6.05 80 OBV Fri Ο, Ε + 00 0, Ι, Ε + 06 3, Ε + 07 6.08 1.44 7.51 81 OBV Fri 2.E + 03 4, E + 04 7.Ε + 04 2, Ε + 06 0.15 1.37 1,52 91 OBV Fri 2.E + 03 Ο, Ε + 00 Ι, Ε + 05 2.Ε + 08 1,62 3.20 4.81 126 OBV Fri 0.E + 00 0, E + 00 7, Ε + 04 Ι, Ε + 06 4.82 1.19 6.01 127 OBV Fri 2.E + 03 0, E + 00 Ι, Ε + 03 Ι, Ε + 06 -0.20 2.97 2.77 130 OBV Fri 0, E + 00 0, E0 3, Ε + 04 2, Ε + 06 4,54 1.80 6.33 176 OBV OBV 0, E + 00 0, E0 2, Ε + 04 Ι, Ε + 06 4.29 1.74 6.03 195 OBV OBV 0, E + 00 0, E0 3, Ε + 06 2, Ε + 08 6.45 1.90 8.34 198 OBV OBV Ι, Ε + 03 0.JEW0 9, Ε + 04 Ι, Ε + 06 1.86 1.14 3.00 205 OBV OBV 0, E + 00 0, UEFA 3, Ε + 04 Ι, Ε + 06 4.41 1,61 6.03 206 OBV OBV 0, E + 00 Ο, ΕΠΗ) 2, Ε + 02 2.Ε + 06 2,30 4.07 6.37 210 OBV OBV 9, E + 03 0, ECU 5.Ε + 03 8.Ε + 06 -0.23 3.19 2.96 216 OBV PT Ο, Ε + 00 Ο, ΕΠΗ) Ι, Ε + 05 4.Ε + 07 4.98 2.61 7.59 217 OBV OBV Ο, Ε + 00 0, E + 00 9, Ε + 03 2, Ε + 06 3.95 2,35 6.30 221 OBV OBV 0, E + 00 Ο, ΕτΰΟ 1, Ε + 05 6, Ε + 06 5.17 1,57 6.74 222 OBV OBV 0.E + 00 0, E + 00 Ι, Ε + 05 2.Ε + 06 5.02 1.28 6.30 232 OBV OBV 0, E + 00 0, E + 00 8.Ε + 05 2, Ε + 07 5.88 1.38 7.27 234 OBV OBV O. E-SCHO 0, Ε + 00 5, Ε + 0Ι 2, Ε + 06 1,69 4.61 6.30 247 OBV OBV 0.E + 00 0, Ε + 00 2, Ε + 04 Ι, Ε + 06 4.37 1.65 6.02 249 OBV OBV 3, E + 04 0, Ε + 00 5, Ε + 04 Ι, Ε + 06 0.27 1.32 1,59 256 OBV OBV 0, E + 00 0, Ε + 00 4.Ε + 04 Ι, Ε + 06 4.60 1.42 6.02 262 OBV OBV 2, E + 03 0, Ε + 00 7.Ε + 06 Ι, Ε + 08 3.60 1.21 4.81 274 OBV Fri 1, E + 04 2, Ε + 04 ό, Ε + 04 Ι, Ε + 06 0.26 1.35 1,61

The second stage included the following.

1. Diagnosis of aerobic vaginitis, based on microscopy of native drugs (vaginal smears) and calculation of aerobic vaginitis points (AV points). The technique for determining AV scores includes an assessment of the lactobacillary degree and morphotypes of concomitant flora, as well as the count of leukocytes and parabasal epithelial cells | Eopbeg8 O.O.O. c1 a1. OGGSHyup OGa 1ure OG aBotta1 Wadsha1 iota 1Pa1 ίδ άίδΐίικ! Goth bac1aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa: VGOS. 2002 1ap; 109 (1): 34-43]. In accordance with the classification proposed by Eopbeg8 S.S.S. e! A1., when receiving 3 AV points or more, the patient is diagnosed with aerobic vaginitis.

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2. The selection of optimal quantitative diagnostic markers of aerobic vaginitis:

a) an assessment of the informativeness of the four main groups of aerobic microorganisms (Eyeg ас bac! e ^^ aseae, B ^ pHu ^^ ss ^ δρρ., B! ^ eρ! οssoci δ δρρ., Е ^ еу ^ сс ^! рр.) showed the unsuitability of E ^ her ^ cc ^ δρρ. as a quantitative diagnostic marker for aerobic vaginitis (Fig. 2);

b) an assessment of the possibility of identifying certain types of aerobic microorganisms for the diagnosis of aerobic vaginitis ^ cNepsyta, B ^ pH ^^ cc ^ angst, B ^ ery ^ cc ^ adaaciae) established a low sensitivity (48%) of this set of markers for the diagnosis of aerobic vaginitis.

Thus, the following three groups of aerobic microorganisms at the level of the genera / families were chosen as quantitative diagnostic markers of aerobic vaginitis: Ejeobieepaseae, B ^ pHu ^^ ss ^ δρρ., B ^ eryu ss ^ δρρ.

3. Determination of the DNA concentration Eyeg ο bac! E ^^ acae, the concentration of bacterial DNA δ ^^ ο ^ οου! δρρ., the concentration of DNA of bacteria BLER ^ otss ^! rr. in a vaginal smear by nucleic acid amplification - multiplex polymerase chain reaction (PCR) with hybridization fluorescence detection in real time.

To conduct a multiplex PCR study from previously obtained vaginal samples, nucleic acids were extracted using a set of reagents for extracting DNA from AmpliPrim DNA-Sorb-AM clinical material (Federal State Budget Research Institute of Epidemiology of Rospotrebnadzor, Moscow, cat. No. 102-22). For further amplification, 10 μl of the obtained eluate containing DNA of the studied microorganisms was used (the volume of the obtained eluate after extraction was 100 μl).

During the preparation of the reaction mixture, a 100-fold TE buffer was used to obtain the necessary concentration of primers, probes, and deoxyribonucleoside triphosphates (άΝΤΡ). The composition of the reaction mixture is given in table. 7.

Table 7

The composition of the reaction mixture (PCR mixture-1)

Title Sequence Fluo rofort Concentration, pmol / μl Primers En (b-P1 §Sv-eSS-SSS-Tv§-ASv-AA§-A 0.7 EPL-K1 §SS-TSA-Av§-§CA-CAA-SST-CCA-A 0.7 ET-Rgo [-K1 Sev-TTS-AAv-ACC-ASA-ACC-TST-AA 0.7 81-e-p eST-ASA-CAC-eTe-STA-CAA-Twe-ASA-A 0.7 81-B-K.1-1 SvT-ATT-SAS-S§T-A @ S-AT§-STv-ATS-TA 0.7 8 (gert-§-G1 VST-ASA-SAS-vtv-STA-SAA-Tvv-TT 0.7 81ger1-§-K.1s CA £ -CST-ASA-ATS-CgA-ACC- £ A £ -ATT 0.6 81ger1-§-rp-K.s CA§-SST-ASA-ATS-SyAA-ACC-§A £ -AST 0.6 8ΤΙ-11 § C _e -T _e C-BAT-8 _§ T-CCё-AST-TAT-T 0.4 8ΤΙ-2 SvS-ASv-TAS-vvS-AAS-TTT-SvT-A 0.4 Probes Εη (6-Ζ2-Ρ (RAM) СёС-ТСО-ВВТ- ₈ Сё-ААА-аСё-Тё £ - е (нн <з RAM 0.2 δι-β-ζυ (K6O) Sayo-SёA-AAS-SёS-ёAё-§TS-AAё- C (CT <3) K6O 0.2 δΙ-Β-δβρΓ-ΖΙ-Ι (CBO) SAV-STA-AAS-СёС-вАв-ВТС-АТв- S (CTF) K6O 0.25 5ΐκρΙ- £ -Ζ1 (-Κ.) (KOKH) SDS-LLE-SS _e - _e T _{g -} LS _e - _e SL-LeS (KTE ·) KOCH 0.2 νΚΟ-Ζ-ΰγ5 (Su5) СТА-ёСТ-в88-С8Т-СА _ё - ₈ AA-TSS-SAё- ₈ (VNfZ) Su5 0.2 Deoxyribonucleoside Triphosphates άΝΤΡ (4 4 tm) 5,0

To obtain the reaction mixture, 10 μl of PCR-mixture-1 and 5 μl of PCR-mixture-2-gey were added to the test tube (FBSI Central Research Institute of Epidemiology of Rospotrebnadzor, Moscow, Cat. No. 863).

Then, 15 μl of the reaction mixture and 10 μl of DNA eluate were added to each amplification tube. In addition, calibrators were used in each formulation for the quantitative analysis of microorganisms (10 μl of calibrators were added to the corresponding tubes). Each formulation involved 2 calibrators with a concentration of 10 and 10 ³ copies / reaction.

- 21 025042

The composition of the calibrators (K1 and K2).

Calibrators include four preparations cloned into plasmids: a concentrated solution, a positive control sample (CR PKO) Ep1goBac1epasee, KR PKO §1arcuosossik krr. For dilution of the initial RS, a DNA buffer with po1u (A) is used. The final concentration of each preparation for the K1 calibrator is 1 · 10 ⁶ copies / ml; for K2 calibrator - 1 -10 ⁴ copies / ml.

Amplification was carried out on a CoYug-Sepa O device (ΟΙΑΟΕΝ, 1011132).

Amplification program:

Ethane Temperature ° C Time Measurement fluorescence Qty cycles But 1 <1 / retention temperature 95 15 minutes - one 1 Susppu / Cycling 95 5 s - 5 60 20 s - 72 15 s - 2 Susp / Cycle 95 5 s - 40 60 20 s RAM, UE, KOH, Su5 72 15s

The results of the study of samples from 22 patients are given in table. 8. For each sample, the following data:

assessment of the state of vaginal microflora according to Amsel’s criteria - bacterial vaginosis (BV) or the absence of bacterial vaginosis (OBV);

assessment of the state of vaginal microflora by Nugent scores - bacterial vaginosis (BV), an intermediate type of microflora (PT) or the absence of bacterial vaginosis (OBV);

bacterial concentrations, expressed in units of GE / ml, determined by real-time multiplex PCR: [DNA Su] - DNA concentration of the bacterium Sagbiege11a uadtaPk, [DNA Αν] - DNA concentration of the bacterium Auropoctus uadtae, [DNA Bas] - DNA concentration of the bacteria crr., [DNA of You] - the concentration of DNA of all bacteria, [DNA Εηΐ] - the concentration of DNA of EXTERNAL aaaaaaaaa, [DNA 5> 1ark | - the concentration of bacterial DNA §1arcu1ossosscr. - the concentration of DNA of bacteria §1ger1ossoss krr .;

the calculated coefficients of ratios of bacterial concentrations: KC1 = 1d [DNA Bas] - 1d ([DNA Su] + [DNA Αν]), KC2 = 1d [DNA Vas] - 1d [DNA Bas], KC3 = 1d [DNA Vas] - 1d ([DNA Su] + [DNA Αν]);

AV points determined by the method of Eopbegg S.S. e! a1.

- 22 025042

Table 8

The results obtained during the examination of 22 patients who have an imbalance in the vaginal microflora, not explained by the presence of

BV-associated microorganisms using the Amsel criteria, Nugent scores, AV scores and real-time multiplex PCR

No. Am spruce New gent and s X 3 > Μ υ D and X 4 and ¢ 9 sa but but Ξ KC1 KC2 KSZ Ε ώ α χ 3 ig ο. 63 55 I 3 Ο. ν and 55 but 3 Ζα ς ϋ Ό Μ nineteen BV Fri 0, E + 00 Ο, Ε + 00 Ι, Ε + 05 Ι, Ε + 06 5.11 1,03 6.14 2, Ε + 03 5, Ε + 05 3.Ε + 06 3 67 OBV Fri 0, E + 00 Ο, Ε + 00 4.Ε + 03 Ι, Ε + 06 3.65 2.40 6.05 3, Ε + 02 8, Ε + Ο3 2, Ε + 06 6 80 OBV Fri 0.E + 00 Ο, Ε + 00 Ι, Ε + 06 3.Ε + 07 6.08 1.44 7.51 7, Ε + 02 Ι, Ε + 07 Ι, Ε + 04 4 81 OBV Fri 2, E + 03 4, Ε + 04 7.Ε + 04 2, Ε + 06 0.15 1.37 1,52 2, Ε + 06 7, Ε + 05 Ι, Ε + 05 3 91 OBV Fri 2, E + 03 Ο, Ε + ΟΟ Ι, Ε + 05 2, Ε + 08 1,62 3.20 4.81 7, Ε + 03 Ι, Ε + 05 Ι, Ε + 08 7 126 OBV PT 0, E + 00 Ο, Ε + 00 7, Ε + 04 Ι, Ε + 06 4.82 1.19 6.01 4, Ε + 05 2, Ε + 04 2, Ε + 06 3 127 OBV PT 2, E + 03 Ο, Ε + 00 1, Ε + 03 1, Ε + 06 -0.20 2.97 2.77 6, Ε + Ο3 Ι, Ε + 04 8, Ε + Ο5 6 130 OBV Fri 0, E + 00 Ο, Ε + 00 3, Ε + 04 2, Ε + 06 4,54 1.80 6.33 3.Ε + Ο3 2, Ε + Ό5 5.Ε + Ο5 5 176 OBV OBV 0, E + 00 Ο, Ε + 00 2, Ε + 04 Ι, Ε + 06 4.29 1.74 6.03 7.Ε + 02 Ι, Ε + 05 2, Ε + 06 4 195 OBV OBV 0, E + 00 Ο, Ε + 00 3, Ε + 06 2.Ε + 08 6.45 1.90 8.34 2, Ε + 03 2, Ε + 03 Ι, Ε + 08 3 198 OBV OBV 1.E + 03 Ο, Ε + 00 9, Ε + 04 Ι, Ε + 06 1.86 1.14 3.00 2, Ε + 06 9, Ε + 04 4.Ε + 05 4 205 OBV OBV 0, E + 00 Ο, Ε + 00 3, Ε + 04 Ι, Ε + 06 4.41 1,61 6.03 4, Ε + 04 2, Ε + 05 3, Ε + Ό6 5 206 OBV OBV 0, E + 00 Ο, Ε + 00 2, Ε + 02 2. Ε + 06 2,30 4.07 6.37 3, Ε + 06 3.Ε + 06 5, Ε + 04 4 210 OBV OBV 9, E + 03 Ο, Ε + ΟΟ 5.Ε + 03 8. Ε + 06 -0.23 3.19 2.96 8.Ε + 03 Ι, Ε + 04 8, Ε + 06 7 216 OBV PT Ο, Ε + 00 Ο, Ε + 00 Ι, Ε + 05 4Ε + 07 4.98 2.61 7.59 5, Ε + 06 Ι, Ε + 07 8, Ε + 03 8 217 OBV OBV Ο, Ε + 00 Ο, Ε + 00 9, Ε + 03 2.Ε + 06 3.95 2,35 6.30 5, Ε + 02 2.Ε + 02 3.Ε + 06 5 221 OBV OBV Ο, Ε + 00 Ο, Ε + 00 Ι, Ε + 05 6. Ε + 06 5.17 1,57 6.74 2, Ε + 06 Ο, Ε + ΟΟ 6, Ε + 02 3 222 OBV OBV Ο, Ε + ΟΟ Ο, Ε + 00 Ι, Ε + 05 2. Ε + 06 5.02 1.28 6.30 2, Ε + 05 Ι, Ε + 06 3, Ε + 05 7 232 OBV OBV Ο, Ε + 00 Ο, Ε + 00 8, Ε + 05 2Ε + 07 5.88 1.38 7.27 3, Ε + 07 Ι, Ε + 06 2, Ε + 06 8 234 OBV OBV Ο, Ε + ΟΟ Ο, Ε + 00 5, Ε + 0Ι 2 Ε + 06 1,69 4.61 6.30 2, Ε + 04 2.Ε + 02 Ι, Ε + 06 8 247 OBV OBV Ο, Ε + ΟΟ Ο, Ε + 00 2, Ε + 04 1, Ε + 06 4.37 1.65 6.02 5.Ε + 04 2, Ε + 06 2.Ε + 05 5 249 OBV OBV 3, Ε + 04 Ο, Ε + 00 5, Ε + 04 1 Ε + 06 0.27 1.32 1,59 3, Ε + 06 ό, Ε + 02 7.Ε + 05 4 256 OBV OBV Ο, Ε + 00 Ο, ΕΗ 00 4, Ε + 04 1 Ε + 06 4.60 1.42 6.02 2, Ε + 02 Ο, Ε + ΟΟ Ι, Ε + 06 Ί 262 OBV OBV 2.Ε + 03 Ο, Ε + 00 7, Ε + 06 Ι, Ε + 08 3.60 1.21 4.81 3.Ε + 08 7.Ε + 03 3, Ε + 06 6 274 OBV PT Ι, Ε + 04 2, Ε + 04 ό, Ε + 04 1 Ε + 06 0.26 1.35 1,61 8.Ε + 04 9, Ε + 05 2.Ε + 03 nine

The calculations confirmed that in all cases of a decrease in the number of lactobacilli in the absence of bacterial vaginosis, lactobacilli were replaced by aerobic microorganisms. In all these cases, aerobic vaginitis was established in patients according to the method of ΌοηάβΓδ O.O.O. c1 a1.

Thus, based on the statistical processing of the obtained experimental data, criteria for differential diagnosis of the absence of imbalance in the vaginal microflora, bacterial vaginosis in combination with an increased content of aerobic microflora and the predominance of aerobic microflora were formed.

With 1d [DNA of You]> 6, ([DNA Εηΐ] + [DNA 5> 1arN] + [DNA §1ger]) <[DNA of Bae] and the absence of bacterial vaginosis, no imbalance of the vaginal microflora is concluded.

When 1d ([DNA Ον] + [DNA Αν]) - 1d ([DNA Εηΐ] + [DNA 5> 1arN] + [DNA 5> 1ger |) <1, the diagnosis of bacterial vaginosis is specified as bacterial vaginosis in combination with an increased content of aerobic microflora.

At 1d [DNA of You]> 5, ([DNA Εηΐ] + [DNA 81ArP | + [DNA 8 £ her])> [DNA Bas], KC2> 1 and KC3> 2 indicate the predominance of aerobic microflora.

For the diagnosis of aerobic vaginitis, patients with an increased content or predominance of aerobic microflora need to additionally assess the presence of a local inflammatory reaction. To do this, determine the presence of clinical signs of inflammation in the patient and the ratio of polymorphonuclear leukocytes: epithelial cells by microscopy of the sample. In the presence of clinical signs of inflammation and the ratio of polymorphonuclear leukocytes: epithelial cells more than 1 diagnose aerobic vaginitis.

Claims (12)

CLAIM 1. A method for the differential diagnosis of imbalances in the vaginal microflora, including determining the concentration of DNA of all bacteria, the concentration of DNA of the bacterium Oypepedea uadsha Pk, the concentration of DNA of the bacterium Aurobium uadtae, and the concentration of DNA of the bacteria Lyophilis bacillus. in the sample by the method of amplification of nucleic acids, characterized in that on the basis of the data obtained, the coefficients of the ratios of the concentrations are calculated: KC1 = 1d [DNA bac] - 1d ([DNA Ον] + [DNA Αν]), KC2 = 1d [DNA of you] - 1d [DNA of bac], KC3 = 1d [DNA of you] - 1d ([DNA Ον] + [DNA Αν]), where [DNA bac] is the concentration of bacterial DNA of Bacillus arr., GE / ml; [DNA Ον] is the concentration of DNA of the bacterium Oagipege 11a uad1pa115. GE / ml; [DNA Αν] is the concentration of DNA of the bacterium Auroptus uadthae, GE / ml; [DNA of you] - DNA concentration of all bacteria, GE / ml; with 1d [DNA of you]> 5, KC1> 1 and KC2 <1, the absence of bacterial vaginosis is noted; when 1d [DNA of you]> 5, 0.5 <KC1 <1, KC2 <1 and KC3 <2, an intermediate state of microflora is observed; with 1d [DNA of you]> 5, KC1 <0.5 and KC3 <2 diagnose bacterial vaginosis; with 1d [DNA of you] <5, it is noted that the number of bacteria is insufficient for analysis. 2. The method according to claim 1, characterized in that in the absence of bacterial vaginosis and 5 <1d [DNA of you] <6, a decrease in the degree of bacterial contamination of the vagina is noted. 3. The method according to claim 1, characterized in that it further determines the concentration of DNA Ep1goLac1epaseae, the concentration of DNA of bacteria §1ръу1ососсий ирр., The concentration of DNA of bacteria 5> 1герУсоссий ррр. in the sample by nucleic acid amplification and 1d [DNA of You]> 6 and ([DNA of Her] + [DNA of 8! Arb] + [DNA of §! Ger]) <[DNA of Bac], where [DNA of Her] is the concentration of DNA of Eeyoebayepaseae, GE / ml; [DNA §1аръ] - DNA concentration of bacteria §1арЬу1ососсий ирр., GE / ml; [DNA §! Ger] - the concentration of bacterial DNA §! Ger! Ossoss yrr., GE / ml, and the absence of bacterial vaginosis conclude that there were no disturbances in the balance of vaginal microflora. 4. The method according to claim 1, characterized in that it further determines the concentration of DNA Ep1goBac1epaseae, the concentration of DNA of bacteria §1ръу1ососсий ирр., The concentration of DNA of bacteria §1per! in the sample by nucleic acid amplification and 1d ([DNA Ον] + [DNA Αν]) - 1d ([DNA Her] + [DNA 8yr] + [DNA 8yr]) <1, where [DNA Her | - the concentration of DNA of Eigoeidepaseae, GE / ml; [DNA §1аръ] - DNA concentration of bacteria §1арЬу1ососси5 рр., ГЭ / ml; [DNA §! Ger] - the concentration of bacterial DNA §! Ger! Ossoss yrr., GE / ml, the diagnosis of bacterial vaginosis is specified as bacterial vaginosis in combination with an increased content of aerobic microflora. 5. The method according to claim 4, characterized in that it further determines the presence of clinical signs of inflammation in the patient and the ratio of polymorphonuclear leukocytes: epithelial cells during microscopy of the sample and in the presence of clinical signs of inflammation in the patient and the ratio of polymorphic nuclear leukocytes: epithelial cells more 1 diagnose bacterial vaginosis in combination with aerobic vaginitis. 6. The method according to claim 1, characterized in that it further determines the concentration of DNA Ep1goBac1epaseae, the concentration of bacterial DNA §1арБу1ососсий ирр., The concentration of bacterial DNA 5> 1герУсоссий ррр. in the sample by nucleic acid amplification and 1d [DNA of You]> 5, ([DNA of Her] + [DNA of 8yp] + [DNA of 8yp])> [DNA of Ba], KC2> 1 and KC3> 2, where [Her DNA] is the concentration of the DNA of Herbillotidae, GE / ml; [DNA §1аръ] - DNA concentration of bacteria §1арЬу1ососсий ирр., GE / ml; [DNA §! Ger] - the concentration of bacterial DNA 5> 1ger1ossoss yrr., GE / ml, ascertain the predominance of aerobic microflora. 7. The method according to claim 6, characterized in that it further determines the presence of clinical signs of inflammation in the patient and the ratio of polymorphonuclear leukocytes: epithelial cells during microscopy of the sample and in the presence of clinical signs of inflammation in the patient and the ratio of polymorphic nuclear leukocytes: epithelial cells more 1 diagnosed with aerobic vaginitis. 8. The method according to claim 1, characterized in that when ascertaining the intermediate state of microflora, the pH of the patient’s secretions and the presence of complaints of pathological discharge in the patient are additionally determined and, at pH <4.5 and the absence of complaints of pathological discharge, the patient is recommended to repeat analysis in the next menstrual cycle, and at pH> 4.5 and the presence of complaints of pathological discharge, the patient is diagnosed with bacterial vaginosis. 9. The method according to claims 1 to 8, characterized in that the sample is a vaginal smear. 10. The method according to claims 1 to 9, characterized in that the method of amplification of nucleic acids is a method of quantitative polymerase chain reaction. 11. The method according to claim 10, characterized in that the method of quantitative polymerase chain reaction is a polymerase chain reaction method with hybridization-fluorescence detection in real time. 12. The method according to claim 10 or 11, characterized in that the polymerase chain reaction is a multiplex polymerase chain reaction. Distribution of patients by the total number of bacteria in the sample (in 1e)