Real-time PCR validation to estimate the number of rickettsias in the biological material under study.

Using the example of the clinical strain of R. sibirica «Bayevo 105/87¼, the possibility of quantitative determination of rickettsias in clinical samples from patients with Siberian tick-borne typhus by real-time polymerase chain reaction (PCR-RT) was evaluated. Cultivation was carried out in the yolk sacs of developing chicken embryos, from which a piece of the yolk sac or chorion was taken. A total of 125 samples were examined. A set of reagents "RealBest DNA Rickettsia species (kit1)" was used for PCR-RT. The obtained values of the threshold amplification cycle (Ct) were compared with the results of microscopy of smear preparations stained by the Zdrodovsky method, the values of which were divided into ranks: the I rank - single rickettsias in individual fields of vision, the II rank - single rickettsias in each field of vision, the III rank - from 10 to 25 rickettsias in each field of vision, the IV rank - from 25 to 50 rickettsias in each field of view. The median Ct value for rank I was 17.6 (16.37; 18.58), for the II - 16.0 (15.0; 16.41), for the III - 15.0 (14.0; 15.1) and for the IV - 15.0 (13.7; 14.64). A significant average correlation was established between the number of rickettsias in the preparation under microscopy and the value of the threshold cycle in PCR RT (r=-0, 4849542; p=9.968e-09). When determining the correlation between the pathomorphological characteristic and the value of the threshold cycle, its absence was established. The detection of rickettsias in the blood vessels of the chorion of developing chicken embryos was of interest. In 10 samples, the yolk sac and chorion were taken for the study, and in parallel they were examined by PCR-RT. The use of modern, more sensitive molecular biological methods allows for quantitative analysis of DNA in the chorion, while preserving the volumes of the most valuable material - the yolk sac.

[Possibilities of serological verification of siberian tick borne type with use of test system for identification of antibodies to Rickettsia conorii.]

The real epidemiological impact of Spotted Fever Group rickettsioses including Siberian tick-borne typhus (STT) in Russia is not sufficiently studied. One of the reasons is the actual absence of either certified domestic diagnostic kits or the evidence for using foreign test kits for laboratory verification of this group of tick-borne infections in medical practice. Objective of our study was to study the diagnostic accuracy of the ELISA test system based on Rickettsia conorii antigens for serological verification of STT. The ROC analysis was performed and operational characteristics (sensitivity, specificity, accuracy, likelihood ratio of positive and negative results) of the STT serological verification test to identify IgM to rickettsia at different times from the onset of the disease using a test system to detect antibodies to Rickettsia conorii were calculated based on the results of a survey of two groups of patients comparable by gender and age (34 patients with pathognomonic signs of STT and 76 clinically healthy people). It was found that the detection of IgM antibodies to rickettsia using the Rickettsia conorii IgM/IgG ELISA test system (Vircell) allows the disease to be verified 10-14 days after the onset of clinical symptoms in 72% (56-88%) of STT patients. We recommend the interpretation of results of the test system "Rickettsia conorii ELISA IgM/IgG" for serological verification of STT which differ from the manufacturer's recommendations regarding verification of Mediterranean fever caused by R. conorii in the following way: the diagnosis of STT should be considered laboratory confirmed when the index of IgM antibodies (IAT) exceeds 8.0; if the IAT is less than 5.0 then a repeated examination of the patient after 10-14 days will be necessary; if the IAT is in the range of 5.0-8.0 then the sample should be re-examined and / or the patient should be examined after 10-14 days. The use of the test system "Rickettsia conorii ELISA IgM / IgG" is promising for laboratory diagnosis and seroepidemiological studies of Spotted Fever Group rickettsioses in Russia.