AREAS OF THE CENTRE’S RESEARCH
1. Ticks identification
Ticks are identified by means of a stereomicroscopic SEO system, which includes a stereomicroscope, a colour digital camera, and a photoadapter.
Types and stages of tick development are distinguished by using special tables.
2. Real-time PCR for the presence of the causative agents’ DNA of the following tick-borne infections: B. burgdorferi s.l. (the causative agent of Lyme borreliosis), B. miyamotoi (the causative agent of the infection caused by B. miyamotoi, A. phagocytophilum (the causative agent of human anaplasmosis), Babesia sp. (the causative agent of human babesiosis) in ticks and human blood.
Research by the PCR makes it possible to detect not only the pathogen itself, but even a single fragment of foreign DNA in the material under study. PCR diagnostics is an accurate and sensitive method of diagnosing infectious diseases, including tick-borne diseases.
The polymerase chain reaction laboratory is equipped with a modern “Rotor Gene 6000” amplifier, which works in the real-time PCR mode, which provides the simultaneous process of amplification and hybridization-fluorescence detection in real time, automatic registration and interpretation of the obtained results.
The CFX96 Touch amplifier (Bio-Rad, USA) is designed for PCR with real-time registration of reaction products. It has a six-channel detection system, which ensures high speed and flexibility of PCR diagnostics.
Real-time PCR has the following advantages:
Quantitative assessment of the genetic material of several infectious agents in dozens of samples.
Prevention of contamination – all stages of analysis take place in one test tube.
Automatic registration of the concentration of reaction products directly during amplification.
The highest sensitivity, versatility and specificity.
The possibility of placing the genetic diagnostics laboratory on one table.
Analysis time reduction.
The laboratory also operates a Perkin Elmer-2400 amplifier, which allows amplification of DNA with subsequent detection by electrophoresis.
3. ELISA (enzyme-linked immunosorbent assay), which allows the determination of total specific IgM and IgG antibodies to the main species of Borrelia burgdorferi s.l.
To carry out this analysis, the Centre has a modern immunoenzyme analyser Multiscan FC.
4. Immunoblot, which allows to determine specific IgM and IgG simultaneously to more than 10 antigens of 4 types of Borrelia burgdorferi s.l.: B. burgdorferi s. s., B. garinii, B. afzelii and B. spielmanii).
The obtained results are evaluated using the EURO Line Scan software, which allows analysing not only the qualitative composition of serum immunoglobulins, but also their quantity.
Figure 1.1 – Sample protocol for evaluation of immunoblot results (EUROLINE Borrelia RN-AT IgM) using EURO Line Scan software
Figure 1.2 – Sample protocol for evaluation of immunoblot results (EUROLINE Borrelia RN-AT IgG) using the EURO Line Scan software
5. Multiplex indirect immunofluorescence using the BIOCHIP (BIOLOGICAL MICROCHIP) technology, which allows simultaneous detection of specific IgM and IgG antibodies to various pathogens of blood infections in the reaction of indirect immunofluorescence, including tick-borne infections (A. phagocytophilum, Borrelia burgdorferi s.l., Bartonella and others).
For this purpose, a matrix with applied molecules of proteins or nucleic acids is used to simultaneously conduct a large number of analyses in one sample.
The obtained results are evaluated by means of a fluorescence microscope Olympus IX70.
A positive sample contains a bright green glow of an antigen-antibody complex labelled with fluorescein.
Рис. 1.
Luminescence of a fluorescein-labelled antigen-antibody immune complex specific for B. henselae. Reactions of indirect immunofluorescence. Olympus IX70 microscope. Patient S., 42 years old. Diagnosis: Localized scleroderma, limited form, chronic course. Anti-IgG to B. henselae is present.
The results of the study are compared with the standard positive and negative controls offered by the manufacturing company.
6. Ultrasound diagnostics of the skin.
Ultrasound diagnostics of the skin is a new and promising direction in the work of dermatologists, surgeons, including plastic surgeons, general practitioners, family doctors and ultrasound specialists.
This method is non-invasive, painless and safe for the life and health of patients, has sufficiently high accuracy, the results are well archived, all types of images can be processed by computer. The studies are performed only in vivo, but without any tissue damage and are suitable for multiple repetitions.
Indications for ultrasound examination in dermatology:
• measurement of the thickness, depth, formation structure and assessment of the borders of skin tumours;
• monitoring the course and therapeutic effectiveness of treatment of diseases with skin sclerosis (scleroderma, panniculitis) and chronic inflammatory dermatoses (psoriasis, lichen planus, and urticaria), including patients with concomitant tick-borne infections (Lyme borreliosis, anaplasmosis, bartonellosis);
• observation after surgery, cryotherapy and laser therapy (malignant melanoma, basal cell carcinoma, intradermal metastases, haemangioma, fibroma, lipoma, seborrheic warts);
• evaluation of exogenous components, such as foreign bodies and cosmetic skin fillers;
• assessment before and after cosmetic injections (localization and condition of the injected drug, places of migration, post-injection compactions and complications).
In their work, the Centre’s employees use the DUB SkinScaner ultrasound device manufactured by Taberna Pro Medicum (Germany) with a 22 MHz sensor.
Photo of stretch marks on the back of patient R., 17 years old. Diagnosis: Bartonellosis, Lyme borreliosis with predominant skin lesions.
The obtained results are interpreted and analysed using the original software for the DUB‑SkinScaner scanner. A standard ultrasonic gel is used as a conductive medium.
Ultrasound image of the focus of the lesion (“stretch marks” of the back): a decrease in the echogenicity of the epidermis. A decrease in the thickness and echogenicity of the dermis (average depth of 760 nm).
Ultrasound image of healthy skin (back area): the thickness of the dermis is 1121 nm.
7. Remote thermography
The method of remote thermography provides information about physiological and pathological processes in the body, thanks to the registration and interpretation of the temperature distribution of the skin of different parts of the human body. It is based on the assessment of temperature differences (ΔT) of affected, symmetrical and intact segments of the human body.
It is relevant to use this method to improve the diagnosis of various clinical manifestations of tick-borne infections, namely Lyme borreliosis (migratory erythema, localized scleroderma, Lyme arthritis, enlarged lymph nodes), anaplasmosis, and bartonellosis.
The main advantages of using remote thermography are the high sensitivity, non-invasiveness, painlessness and non-contact of this method, the ability to quickly carry out it and objectively evaluate the images.
We conduct the research with a medical thermal imager ULIRVISION T1-120.
Thermographic picture of migratory erythema of the left lower leg of a Lyme borreliosis patient, front view (focal heating), patient G., 43 years old.
To analyse thermographic images, we use the “IRSee Software” software package.