Contemporary researchers estimate that over 357 million people a year contract bacterial pathogens of sexually transmitted infections (STIs) [1, 2], and the frequency of inflammatory diseases of urogenital tract caused by aerobic and anaerobic opportunistic microorganisms reaches 80% among genital sphere pathologies [3-5].
With the aim to reduce incidence of and eliminate STIs there was developed the Global Health Sector Strategy on Sexually Transmitted Infections (2009–2016, 2016–2021). One of the missions pursued by the strategy is popularization of early diagnostics of such diseases, including symptom-free cases, since diagnosing an STI at an early stage creates optimal conditions for effective treatment and helps prevent further spread of the infectious agents [2].
Until recently, culture test was the “golden standard” in urogenital infections diagnostics. However, a significant number of etiological agents of urogenital tract infections and inflammations are hard to culture or unculturable. Besides, culture testing is labor intensive and time consuming, which significantly limits its use in routine clinical practice. Thus, there is a need for new diagnostic technology for urogenital pathology.

Diagnostic methods for urogenital infections

The current recommendation is to perform extra tests to confirm gonococcal infection diagnosed through microscopic examination only if the patient is male and the disease manifests itself. Microscopy has a number of deficiencies that limit its applicability significantly: the assessments of results are subjective, the sensitivity is low (30–40%) when examining cervical, pharyngeal and rectal samples, as well as in cases of asymptomatic infections [6]. For children, pregnant women, women during menopause and in suspected cases of extragenital and complicated forms of the disease it is necessary to use culture and/or molecular biological testing methods. In N. gonorrhoeae identification, the specificity and sensitivity offered by molecular biological methods reaches 100%. They are given priority in the routines of diagnosing asymptomatic forms of the disease, when a mixed infection is suspected, in screening examinations and when there are anamnestic and/or clinical signs of gonococcal infection with negative results of microscopic examination or when there are altered gram-negative or gram-stable diplococci detected [7, :lit_8 ;].

The basic methods of diagnosing urogenital trichomoniasis are native preparation microscopy, molecular biological and culture methods. Native preparation microscopy is performed immediately after sampling biological material, which limits applicability of this method. Microscopy of stained preparations is not recommended for diagnostic purposes since its sensitivity and specificity are the lowest (30–60%) compared to other laboratory methods (Trichomonas vaginalis are often round and resemble polymorphonuclear leukocytes, and its typical morphological signs are lost during fixation and staining) [6].
Culture method's sensitivity and specificity largely depend on the composition of culture media and conditions of Trichomonas culturing. This method is more labor intensive and time consuming than molecular biological methods, the sensitivity of which is 88–97% and specificity — 98–99%. Molecular biological testing is given priority in cases of asymptomatic trichomoniasis (most often observed in males), when there is a mixed infection suspected, in screening examinations, and also when there is a need to control the quality of microscopic examination [5, 8].
It is recommended to apply only the molecular biological methods, with their specificity and sensitivity approaching 100% [5, 9], to validate diagnosis of chlamydial infection, diseases caused by M. genitalium, and viral STIs (anogenital herpes virus and human papillomavirus infections).
Thus, molecular biological methods — polymerase chain reaction (PCR), real-time PCR (RT PCR), NASBA — were added to the official lists as methods to diagnose all sexually transmitted infections.

Possibilities offered by molecular biological methods in diagnosing urogenital system pathologies

Introduction of PCR to clinical practice had a truly revolutionary effect on identification of STIs agents. However, improperly selected genetic targets and/or various inhibitory factors can affect sensitivity of PCR. Moreover, its specificity may also be hindered by non-pathogenic representatives of the same genus/family of microorganisms present in the sample. Besides, qualitative PCR does not provide sufficient information to identify opportunistic microorganisms.
Real-time PCR is a modification of PCR that combines amplification and simultaneous detection of accumulation of its products directly during the reaction. This method allows not only to determine DNA of the microorganism but also quantify its content in the sample. Real-time PCR does not involve post-amplification analysis of reaction products and extraction of the test tube contents, which significantly lowers the risk of sample contamination and eliminates the need for a separate laboratory zone, as well as reduces testing time and augments objectivity of interpretation of its results [10].
Until recently, only the culture method allowed identification of opportunistic aerobic microorganisms, while anaerobic microorganisms, which often cause inflammatory and dysbiotic diseases of genitourinary system, remained undetected. Meanwhile, it is known that this group of bacteria often competes with lactobacilli for biotope dominance, and with dysbiotic disorders in women, it is usually obligate-anaerobic microorganisms that colonize the vaginal epithelium [11].

Today, real-time PCR enables adequate diagnostics of STIs and simultaneous detection of dysbiotic conditions (aerobic/anaerobic vaginitis, urethritis, balanoposthitis, bacterial vaginosis, etc.) resulting from an imbalance between opportunistic microorganisms and normal urinary tract microbiota. There is a number of testing systems based on this method (Florocenosis, Ampliflor, Amplisens etc.) registered in Russia; they allow qualitative and quantitative assessment of pathogenic and opportunistic aerobic microorganisms. Florocenosis and Ampliflor enable identification of lactobacilli, representatives of the Candida genus, family Enterobacteriaceae, Streptococcus spp., Staphylococcus spp., G.vaginalis, A.vaginae, opportunistic mycoplasmas, but they do not allow detecting obligate anaerobic microorganisms. Femoflor-16 and Androflor testing systems offer the widest detected range of opportunistic aerobic and anaerobic microorganisms of all the systems available. Application of these unique “paired” tests, developed for women and men, allows developing an effective algorithm for laboratory examination of couples and selection of therapy if one or both partners have infectious diseases of reproductive system or reproductive function disorders (fig. 1, fig. 2) [12, 13].
From the clinical point of view, one of the most important features of these testing systems is sampling quality control: the marker of sufficiency of collected epithelial cells that enter transport medium when scraping. At the early stages of examination, this indicator allows assessing adequacy of biomaterial sampling and avoiding false positive and false negative results [14, 15].

Absolute quantitative results of real-time PCR are given in genome equivalents (GE), the values of which are proportional to microbial contamination of urogenital biotope. However, according to some researchers, absolute quantitative indicators do not always correlate with severity of clinical picture of the disease, and values in excess of their thresholds do not always signal of clinical manifestations of an infectious-inflammatory process. Back in 1986, F. J. Roberts found a bacteriuria level below significancy in 30% of patients with clinically and laboratory confirmed bacterial infections of genitourinary tract, and in 1987 J. A. Kellogg et al. found no correlation between clinical manifestations of urethritis and a significant level of bacteriuria. In this connection, determining the relative quantitative indicators (shares) of opportunistic microorganisms as a logarithmic difference and as a percentage of the total bacterial mass is of great practical interest. This indicator allows a specialist to assess the share of each microorganism or group of microorganisms in a specific biological sample, as well as evaluate their prevalence over other species, including normoflora, which significantly increases clinical significance of the test.

CONCLUSIONS

PCR, previously discredited as oversensitive for urogenital pathology diagnosing, justifies itself through introduction of quantitative PCR tests into routine clinical practice, which enable express testing providing a comprehensive assessment of the state of urogenital biocenosis. This approach demonstrates the ratio of key representatives of the microbial community and reduces the share of false positive and false negative results.