Evaluation of serological indicators of the presence of antibodies to pathogens of natural focal infections in the population of the Rostov region in 2021

Introduction

An important indicator confirming the presence of pathogens of natural focal infections (NFIs) in a certain area is the availability of specific antibodies in the blood sera of people living in this region [1]. Revealing seropositive individuals makes it possible to earlier detect the circulation of a particular infectious agent, forecast the epidemiological situation, and prevent epidemic complications since antibodies are a unique biomarker for identifying people with protective immunity. Based on the examination results of the immune layer level among the population of a certain territory, a reliable conclusion can be made about the degree of natural foci activity and the pathogen prevalence [2][3]. Serological investigations are a direct and informative method available for determining the dynamics of population susceptibility to diseases and assessing the population immunity level [4].

Natural and climatic factors, as well as landscape and geographical zones, in the Rostov Region (RR) determine the appearance of territories with generated persistent natural foci of such dangerous infectious diseases as tularemia, West Nile fever (WNF), Crimean hemorrhagic fever (CHF), ixodid tick-borne borreliosis (ITBB), and some others diseases [5][6]. In recent years, in the RR, the largest proportion (88.51%) of registered transmissible NFIs has accounted for CHF, WNF, and ITBB¹. Currently, 42 administrative territories of the RR are endemic for CHF and there is a tendency to further expand their verges [7]. In 2021, 16 laboratory-confirmed cases of CHF were registered². According to the Rospotrebnadzor for the RR, in 2021 there were two cases of WNF [8]; five cases of ITBB were confirmed in Rostov-on-Don, Kamensk-Shakhtinsky, and Orlovsky districts³.

Currently, the RR refers to subjects with a low incidence of hemorrhagic fevers with renal syndrome (HFRS). The first laboratory-confirmed case of the disease was registered in 2018 in the Peschanokopsky district, and then in 2019 one patient was detected in the Salsky district. For 2020–2021, there are no official data on cases of registration of HFRS in the RR.

The antigen of the HFRS pathogen was detected during epizootological monitoring from samples of biological material of small mammals in 2019, which confirms the circulation of the HFRS pathogen on the RR territory [6]. In 2020, the investigation of the immune layer among the healthy population revealed class G antibodies among 6.8% of residents. The registration of virus markers in samples of carriers and the presence of a natural immune layer of the population in relation to HFRS pathogen viruses indicate the demand for epidemiological surveillance for this infection [9].

Natural foci of tularemia of the steppe and floodplain-marsh types are located in 35 out of 42 administrative districts of the RR. The periodic activity of infection foci has been recorded in these territories for many years. Over a five-year period from 2017 to 2021, Francisella tularensis cultures were isolated in several districts of the RR, namely, Tselinsky in 2017, Salsky and Remontnensky in 2020 [10][11]. Along with the isolation of cultures of the tularemia causative agent in 2017, five cases of disease were detected among the population of the region [11].

The purpose of the study was to analyze the results of a comprehensive investigation of blood sera among the local population of the RR conducted in 2021 to assess the state of natural and anthropurgic foci of CHF, WNF, ITBB, and tularemia, as well as the possible risk degree of HFRS spreading.

Materials and methods

The study included blood sera obtained from healthy donors living in various administrative territories of the RR in 2021. The biomaterial was obtained in accordance with the principles of legality and ethical standards. Informed consent was obtained from each donor. The selection took into account the permanent residence of donors in the territories of natural foci revealed by epizootological monitoring and the absence of a previous history of NFIs.

Serological monitoring included the investigation of a wide range of antibodies to pathogens of the examined infections in each individual sample.

Specific antibodies in donor blood samples to the pathogens of CHF, WNF, ITBB, and HFRS were determined by ELISA. Test systems manufactured by JSC "Vector-Best" (Novosibirsk), "VektoKrym-KHL" (IgM, IgG), "VektoNil" (IgM, IgG), "LimeBest" (IgM, IgG), and "VektoHanta" (IgM, IgG) were used according to the manufacturer's instructions. The reaction results were read by an Infinite F50 recording photometer (Tecan, Austria)

Antitularemia antibodies were detected in the system of reactions IHAR/IHAIR (indirect hemagglutination reaction/indirect hemagglutination inhibition reaction) with antigenic liquid erythrocyte tularemia diagnosticum (IHAR-Tul-Ag-StavNIPCHI). The investigation was carried out in accordance with methodological instructions MU 3.1.2007-05 "Epidemiological surveillance of tularemia" and methodological instructions MUK 4.2.2939-11 "The procedure for organizing and conducting laboratory diagnostics of tularemia for laboratories of the territorial, regional and federal levels”. Antibody titers ≥ 1:40 with confirmation of specificity in IHAIR were taken as a positive result.

Confidence intervals for the proportion of positive samples were determined by the Wilson method with a confidence probability of p³0.95; comparison of the proportions of seropositive sera was carried out on the base of the chi-square test with a confidence probability of p³0.95 using software tools provided by the website https://epitools.ausvet.com.au.

Results

The results of a screening investigation of 424 blood sera conducted in 2021 among healthy donors living in 10 administrative territories (Rostov-on-Don, Shakhty, Kamensk-Shakhtinsky, Volgodonsk, Taganrog, Zernograd, Morozovsk and Salsky, Neklinovsky, and Azov districts) are presented in Table 1.

Isolated detection cases of class G immunoglobulins to the Crimean-Congo hemorrhagic fever virus (CCHF virus) were registered in Taganrog and Zernograd. Overall, the proportion of seropositive results was 0.7%.

Antibodies to the West Nile virus (WNV) were detected in all the examined territories, namely, in towns of Morozovsk 20.6%, Volgodonsk 10.0%, Taganrog 8.2%, Shakhty 8.9%, Rostov-on-Don 5.8%, Kamensk-Shakhtinsky 5.6%, Zernograd 5.0%, as well as in two districts of the RR (Salsky 8.6% and Neklinovsky 17.1%). One resident of Shakhty town, a 68-year-old man, was simultaneously found to have antibodies of classes M and G. The highest percentage of positive results (69.4%) for WNV was obtained among people of the older age group (50–77 years), most of whom were women.

In a seroepidemiological examination of healthy donors for the presence of antibodies to ITBB pathogens, class G immunoglobulins were found in the blood of 2.6% of the people. The largest number of IgG-positive results was registered in the Neklinovsky district (8.6%), Rostov-on-Don (5.9%), and Morozovsk (5.9%). Specific immunoglobulins of classes M and G were simultaneously detected in one sample from a resident of Shakhty town and in one sample from a resident of Kamensk-Shakhtinsky town. The analysis of the results showed that 63.6% of seropositive samples belonged to men in the age group of 55 to 72 years.

Virus-specific IgGs to HFRS were detected in the towns of Taganrog (12.2%), Morozovsk (11.8%), Mines (5.4%), and Zernograd (5.0%); also, isolated cases were recorded in Rostov-on-Don, Kamensk-Shakhtinsky, and Volgodonsk. Wherein, it should be noted that there were no registered cases of the disease on the territory of the RR in 2021. The median age of seropositive donors was 54 years. A 20-year-old woman from Zernograd and a 60-year-old man from Morozovsk had simultaneously antibodies of classes M and G, which suggests a recent contact of these donors with the pathogen and, apparently, its active circulation within these territories. The average frequency of detection of seropositive tests for HFRS was 5.1%.

The investigation of the sera of RR residents for the presence of specific anti-tularemia immunoglobulins showed that in different areas, the proportion of positive results varied within 5.9–54.3%. The highest percentage was found among the rural population in the Salsky (54.3%) and Azov (50.0%) districts, as well as among the residents of Zernograd (40.0%). In large towns, the proportion of positive samples was lower and amounted to 10.0% in Volgodonsk, 11.1% in Kamensk-Shakhtinsky, 16.1% in Shakhty, 16.3% in Taganrog, and 19.6% in Rostov-on-Don. The median age of seropositive donors was 50.5 years.

The results of the conducted monitoring are included in the database "Serological monitoring of NFIs in the Rostov Region" (Certificate of state registration No. 2020621999 dated October 22, 2020). This made it possible to compare the results obtained in 2020 and in 2021 (Fig. 1).

The data obtained for two years demonstrate the presence of an immune layer among the population of the RR to viruses of CCHF, WNV, HFRS, pathogens of ITBB and tularemia. Differences in the proportions of seropositive samples in 2020 and 2021 are not statistically significant.

Discussion

Immunological screening is an important diagnostic tool for determining epidemiological diagnosis in the epidemiological surveillance system [12]. Simultaneous measurement of the antibody level to several pathogens, in addition to information about the true structure of the infectious morbidity in the region and circulating pathogens, can identify population groups with a high probability of being infected with several infectious agents simultaneously [13]. Comparing the results obtained during seromonitoring of the RR population with the epizootological and epidemiological situation for these infections, one can conclude about circulating causative agents of CHF, WNVF, ITBB, HFRS, and tularemia, as well the as high frequency of contacts of the local population with these etiological agents.

Crimean hemorrhagic fever. In 2021, the detection rates for specific antibodies in the examined territories were 0.7%. Against the background of regular laboratory-confirmed cases of CHF among the RR residents, the investigation of the immune structure of the population revealed low rates of seropositivity. Similar results were obtained during serological monitoring in 2009–2010 [14]. The low detection rate of specific antibodies to the CCHF virus may be determined by several factors including a sharp decrease in the level of class G immunoglobulins in convalescents by the end of the first year [15] and, probably, by the random nature of the donor selection, not covering high-risk groups (those living in rural areas and those working in animal husbandry and agricultural enterprises). The results of the conducted work allow concluding about the demand of expanding both the geography of the investigation and the range of professional affiliation of the tested.

West Nile fever. The proportion of seropositive results was 9.9%. The IgG availability in the blood serum of local residents to the WNF virus indicates its circulation within the territory of the region subjects. Conducted from 2008 to 2016 laboratory investigations of the healthy donor sera for the availability of antibodies to the West Nile virus among the population of the Salsky, Oktyabrsky, Kamensky districts and the town of Taganrog showed that positive results were identified throughout the monitoring period, with the percentage of positive samples ranging from 0.3% (2016) to 8.5% (2014). Most of the seropositive donors were revealed in the Salsky district [6]. In 2020, cases of WNF were not registered, which may be caused by restrictive measures for a new coronavirus infection, however, samples seropositive for WNV were detected in towns Rostov-on-Don, Kamensk-Shakhtinsky, and Volgodonsk, as well as in Salsky, Remontnensky, and Neklinovsky districts (IgG was registered in 8.7% of cases) [9]. In 2021, two cases of the disease were identified [8]; while in this investigation, the proportion of seropositive findings for WNV was 9.7%. Taking into consideration the fact that up to 80% of cases are asymptomatic or with mild symptoms, it can be assumed that not all cases of the disease are revealed by medical organizations in the region.

Ixodid tick-borne borreliosis. In 2021, according to the Rospotrebnadzor data, 5 cases of ITBB disease were registered, namely, in Kamensk-Shakhtinsky (1), Rostov-on-Don (3), and the Orlovsky district (1), which confirms the activity of the natural focus in the RR [On the state of sanitary epidemiological well-being of the population in the Russian Federation in 2021: State report. Moscow: Federal Service for Supervision of Consumer Rights Protection and Human Welfare, 2022]. In the present investigation, antibodies to ITBB were found in 2.6% of cases.

Hemorrhagic fever with renal syndrome. The data available in the literature indicate that natural foci of HFRS are located in the territories verged on the RR. The circulation of HFRS viruses was confirmed in Kalmykia, Volgograd Region, Stavropol and Krasnodar Territories, and Ukraine [16][17]. The proportion of positive samples for HFRS in this investigation was 5.1%. There is a discrepancy between the low registration level of the Hantavirus infection incidence in the region and the detection of specific antibodies in the population of the RR. This may be stipulated by the dominance of light undiagnosed forms of the disease, without a pronounced hemorrhagic syndrome. In this regard, the infection demands close attention and constant monitoring for the activity of possible natural foci.

Tularemia. According to the literature, during the serological screening of random samples among the population, seroprevalence for Francisella tularensis ranges from 0.19% to 4.0%, while in the territories of natural foci or among special contingents with an increased professional risk of infection (hunters, fishermen, agricultural workers, etc.), this index ranges from 5.0% to 18.0% [18]. In the present investigation, a high percentage of seropositive donors (in the Salsk, Azov districts and the town of Zernograd) is apparently stipulated not only by the circulation of the pathogen in active tularemia foci but also the fact that tularemia is a vaccine-controlled infection, and the examined group by random sampling may have included people previously vaccinated against tularemia. Most districts of the region are endemic in terms of the infection, and in accordance with the current documents, routine population immunization with a live tularemia vaccine is put into practice on the territory of the RR for specific disease prevention. Wherein, antibodies against F. tularensis in immunized people, as well as in recovered patients, can persist for a long time (up to 20 years) [19]. According to regulatory documents (MU 3.1.2007-05 "Epidemiological Surveillance of Tularemia"), the immune layer of the population for a floodplain-swamp type focus should be ≥ 90%, while for a meadow-field type focus ≥ 70%. It cannot be ruled out that the reduced indices of the immune layer obtained for some endemic regions may be caused by the negative attitude of selected people groups toward vaccination.

For a further more correct assessment of the pathogen circulation in the focus, it is advisable to analyze a special sample of people who have not previously been vaccinated with the tularemia vaccine. Along with this, it is demanded to conduct an in-depth test of the intensity of anti-tularemia immunity in the vaccinated population in order to increase the effectiveness of preventive measures in natural foci of tularemia in the RR.

Conclusion

The results of monitoring demonstrate extensive, mostly combined nosoareas of NFIs on the RR territory, confirming the circulation of pathogens of viral and bacterial etiology and showing a high frequency of contacts of the local population with them. It is relevant to conduct regular immunological screening as a diagnostic tool for determining an epidemiological diagnosis in the system of epidemiological surveillance.