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QUANTITATIVE PCR IN DIAGNOSING INFECTIOUS UROGENITAL PATHOLOGY
Rakhmatulina MR1 Galkina IS2
1 Burnasyan Federal Medical Biophysical Center of Federal Medical Biological Agency, Moscow, Russia
2 Federal Research Institute for Health Organization and Informatics of Ministry of Health of the Russian Federation, Moscow, Russia
This article describes the contemporary methods of diagnosing sexually transmitted infections, their advantages and disadvantages, indications for use. The authors describe application of quantitative polymerase chain reaction in diagnosing inflammatory diseases and dysbiotic conditions in men and women. This method, which is currently the "golden standard" in urogenital pathology diagnostics, has undeniable advantages over microbiological methods and qualitative polymerase chain reaction: the preanalytical stage requirements (preservation of quantitative ratios between microorganisms or nucleic acids of microorganisms) are not as strict, the risk of contamination from outside environment and subsequent corruption of the results is significantly smaller, the conditions for all microorganisms, including those impossible and hard to cultivate, are the same sensitivity and specificity-wise, it is possible to sample materials and evaluate microbiota (ratios of microorganisms and their groups) and also possible to collect samples non-invasively, the speed of testing is high. Keywords: microbial biocenosis, urogenital infections, qPCR
Declarations: the authors declare a conflict of interests in connection with DNA-Technology JSC, developer and manufacturer of the reagents mentioned in the article.
Author contribution: Rakhmatulina MR — literature data analysis, text authoring and editing; Galkina IS — literature data analysis, text authoring.
Correspondence should be addressed: Margarita R. Rakhmatulina Novoyasenevsky Prospekt, 9, Moscow, 117588; [email protected]
Received: 03.12.2019 Accepted: 18.12.2019 Published online: 25.12.2019
DOI: 10.24075/brsmu.2019.088
ДИАГНОСТИКА ИНФЕКЦИОННОЙ УРОГЕНИТАЛЬНОЙ ПАТОЛОГИИ МЕТОДОМ КОЛИЧЕСТВЕННОЙ ПЦР
М. Р. Рахматулина1 И. С. Галкина2
1 Федеральный медицинский биофизический центр имени А. И. Бурназяна, Москва, Россия
2 Центральный научно-исследовательский институт организации и информатизации здравоохранения Минздрава России, Москва, Россия
В статье представлены современные методы диагностики инфекций, передаваемых половым путем, их преимущества и недостатки, показания к применению. Описаны возможности количественной полимеразной цепной реакции для диагностики воспалительных заболеваний и дисбиотических состояний у мужчин и женщин. Данный метод, являющийся в настоящее время «золотым стандартом» диагностики урогенитальной патологии, имеет неоспоримые преимущества перед микробиологическими методами и полимеразной цепной реакцией в качественном формате: менее жесткие требования к преаналитическому этапу для сохранения количественных соотношений между микроорганизмами (нуклеиновыми кислотами микроорганизмов), значительно меньший риск влияния на результат исследования контаминации образцов микроорганизмами из внешней среды, равные условия по чувствительности и специфичности для всех микроорганизмов, в том числе некультивируемых и труднокультивируемых, возможность контроля взятия материала и оценки состояния микробиоты по отношению микроорганизмов и их групп друг к другу, скорость получения результата и возможность исследования неинвазивно взятых образцов.
Ключевые слова: микробный биоценоз, урогенитальные инфекции, количественная полимеразная цепная реакция
Декларации: авторы заявляют о наличии конфликта интересов в связи с аффилиацией с группой компаний «ДНК-Технология» — разработчиком и производителем упоминаемых в статье реагентов.
Информация о вкладе авторов: М. Р. Рахматулина — анализ данных литературы, написание и редактирование текста; И. С. Галкина — анализ данных литературы, написание текста.
[23 Для корреспонденции: Маргарита Рафиковна Рахматулина
Новоясеневский проспект, д. 9, г. Москва, 117588; [email protected]
Статья получена: 03.12.2019 Статья принята к печати: 18.12.2019 Опубликована онлайн: 25.12.2019 DOI: 10.24075/vrgmu.2019.088
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 symptomfree 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, 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).
Biomaterial sample
I
Pronounced decrease of normal flora
PRONOUNCED DYSBIOSIS
Moderate decrease of normal flora
MODERATE DYSBIOSIS
I Quantity of normal flora ▼ not decreased
NORMOCENOSIS
OPPORTUNISTIC MICROFLORA EVALUATION
Estimation of share of aerobic bacteria
Above threshold
Estimation of share of anaerobic bacteria
Above threshold
Estimated quantity of Candida spp., Ureaplasma spp. and Mycoplasma hominis
Above threshold
Below threshold
(Aerobic dysbiosis ) i Mixed dysbiosis ) ( Anaerobic dysbiosis ) Cton^foral f Absolute \
normocenosis normocenosis
DYSBIOSIS ETIOLOGY
Decision on the need for sanation
Decision on etiotropic therapy
Fig. 1. The basic algorithm for interpreting urogenital tract microflora examination, female patients, real-time PCR
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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, 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
Biomaterial sample
i
Abbreviations: TBM — total bacterial mass, hgDNA — human genomic DNA, LB — lactobacilli, OM — opportunistic microorganisms, TH — threshold value
Fig. 2. The basic algorithm for interpreting urogenital tract microflora examination, male patients, real-time PCR
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].
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.
References
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Литература
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