Evidence of tick-borne encephalitis virus neutralizing antibodies in Serbian individuals exposed to tick bites.

Introduction: Tick-borne encephalitis (TBE) is an emerging vector-borne and food-borne disease caused by the tick-borne encephalitis virus (TBEV; Orthoflavivirus encephalitidis), with a distribution spanning the Eurasian continent. Despite its significant public health impact in various European regions, TBE remains largely underdiagnosed in Serbia due to limited awareness and diagnostic challenges. In response to this, our study aimed to comprehensively assess TBEV exposure in individuals infested with ticks and to identify potential TBEV foci within Serbia. Materials and methods: From 2019 to 2021, we conducted an observational study involving 450 patients who reported tick infestations. Results: Our demographic analysis revealed a median age of 38 years, with a slight male predominance among the participants. We documented tick infestations in 38 municipalities across 14 districts of Serbia, with a notable concentration in proximity to Fruska Gora Mountain. The ticks most frequently removed were Ixodes ricinus, with nymphs and adult females being the predominant stages. On average, nymphs were removed after about 27.1 hours of feeding, while adult females remained attached for approximately 44.4 hours. Notably, we found age as a significant predictor of infestation time for both nymphs and adult females. Furthermore, we detected TBEV-neutralizing antibodies in 0.66% of the serum samples, shedding light on potential TBEV foci, particularly in Fruska Gora Mountain and other regions of Serbia. Conclusion: Our study emphasizes the urgent need for active TBE surveillance programs, especially in areas suspected of hosting TBEV foci, in order to assess the true TBE burden, identify at-risk populations, and implement effective preventive measures.

Tick-Borne Encephalitis Virus and Borrelia burgdorferi Seroprevalence in Balkan Tick-Infested Individuals: A Two-Centre Study.

Lyme borreliosis (LB) and tick-borne encephalitis (TBE) are important tick-borne diseases in Europe. This study aimed to investigate the seroreactivity against Borrelia burgdorferi and TBE virus (TBEV) in tick-infested individuals in North Macedonia and Serbia. Serum samples were collected from tick-infested individuals and from healthy individuals in the same regions. Samples were tested for anti-Borrelia IgG reactivity and TBEV-neutralizing antibodies. Results showed higher seroreactivity against Borrelia antigens in patients and healthy donors from Novi Sad compared to those from the Skopje region. However, there was no statistically significant difference between tick-infested patients and healthy donors within each region. No TBEV-neutralizing antibodies were detected in participants from Novi Sad or in the control groups, except for one person from North Macedonia who had a moderate TBEV-neutralizing reaction. The study highlights the need for improved surveillance and diagnostic capabilities for LB and TBE in these regions. It also suggests the potential existence of TBEV foci in North Macedonia. The findings provide a complementary understanding of the LB and TBE epidemiology in the studied regions; however, further research is needed to investigate the presence and distribution of Borrelia spp. and TBEV in ticks to assess the significance of detected seroreactivity.

Emerging tick-borne spotted fever group rickettsioses in the Balkans.

The impact of tick-borne pathogens (TBPs) on human health has increased in the last decades, since the incidence of emerging and re-emerging infectious and zoonotic tick-borne diseases has increased worldwide. Tick-borne rickettsiae of the Spotted Fever group (SFGR) are considered as emerging pathogens that can infect humans and cause a variety of non-specific clinical symptoms. Here, we report nine cases of atypical tick-borne diseases (9/460; 1.95%) that occurred over a period of four months (from 15 April 2021 to 16 August 2021) in Serbia, from which five cases were classified as confirmed SFGR infection, two cases as probable SFGR infection and two cases as suspected SFGR infection. Within cases of confirmed SFGR infection, R. helvetica was detected as the causative agent in two cases. The most common clinical finding was non-expanding persistent circular redness, followed by eschar and enlargement of regional lymph nodes, and pain at lesion site. Rickettsia outer membrane protein B (ompB) and citrate synthase (gltA) gene fragments were amplified from clinical samples and ticks attached to patients and IgG reacting with Rickettsia conorii antigen were detected in sera samples of patients, which are highly suggestive of exposure to SFGR. Surveillance and monitoring of rickettsial diseases in Serbia should continue and extended to new areas due to the increasing trend of clinical infections caused by SFGR in the country.

Differential detection of tick-borne pathogens in human platelets and whole blood using microfluidic PCR.

The tick-borne pathogens (TBPs) with adhesive phenotype can use platelets for dissemination and colonization of distant tissues and organs, and it has been shown that they can be found concentrated in the platelet fraction of blood. This study shows the differential presence of TBPs in samples of human platelet fraction (n = 68), whole blood samples (n = 68) and ticks collected (n = 76) from the same individuals, using an unbiased high-throughput pathogen detection microfluidic system. The clinical symptoms were characterized in enrolled patients. In patients with suspected TBP infection, serological assays were conducted to test for the presence of antibodies against specific TBPs. Tick species infesting humans were identified as Ixodes ricinus, Dermacentor reticulatus, and Haemaphysalis punctata. Eight patients developed local skin lesions at the site of the tick bite including non-specific lesions, itching sensation at the lesion site, and eschar. Most common TBPs detected in platelet fraction were Borrelia spielmanii and Rickettsia sp., followed by Borrelia afzelii and Anaplasma phagocytophilum. Multiple infections with three TBPs were detected in platelet fraction. In whole blood, most common TBPs detected were Anaplasma spp. and A. phagocytophilum, followed by Rickettsia spp. and B. afzelii. In ticks, the most common TBP detected was Rickettsia spp., followed by Borrelia spp. and Anaplasma spp. Overall, nine different pathogens with variable prevalence were identified using species-specific primers, and the most common was Rickettsia helvetica. In three patients, there were no coincidences between the TBPs detected in whole blood and tick samples. Only in one patient was detected A. phagocytophilum in both, whole blood and tick samples. These results suggest the unequal detection of TBPs in whole blood, platelet fraction and ticks collected, from the same individual. The results justify the use of both whole blood and platelet fraction for molecular diagnosis of TBPs in patients.

Fatal Case of Imported Tick-Borne Encephalitis in South Serbia.

Tick-borne encephalitis (TBE) is vaccine-preventable neglected zoonotic neuroinvasive disease, caused by tick-borne encephalitis virus (TBEV). Many of the Central and Eastern European countries are affected by TBE, which is often poorly perceived by tourists visiting endemic territories. Here we are reporting a fatal case of imported TBE in Serbian resident who was exposed to a tick bite during a visit to Switzerland.

Mediterranean spotted fever-like illness caused by Rickettsia sibirica mongolitimonae, North Macedonia, June 2022.

Mediterranean spotted fever-like illness (MSF-like illness) is a tick-borne disease caused by Rickettsia sibirica mongolitimonae first reported in France more than 25 years ago. Until today, more than 50 cases of MSF-like illness have been reported in different regions of Europe and Africa, highlighting variable clinical manifestation. Here we report a case of MSF-like illness following a bite from a Hyalomma tick in the Skopje region of North Macedonia.

Unexpected TBEV Seropositivity in Serbian Patients Who Recovered from Viral Meningitis and Encephalitis.

The tick-borne encephalitis virus (TBEV) causes a life-threatening disease named Tick-borne encephalitis (TBE). The clinical symptoms associated with TBE range from non-specific to severe inflammation of the central nervous system and are very similar to the clinical presentation of other viral meningitis/encephalitis. In consequence, TBE is often misclassified by clinical physicians, mainly in the non-identified high-risk areas where none or only a few TBE cases have been reported. Considering this situation, we hypothesized that among persons from northern Serbia who recovered from viral meningitis or encephalitis, there would be evidence of TBEV infection. To test this hypothesis, in this observational study, we evaluated the seroreactivity against TBEV antigens in patients from northern Serbia who were hospitalized due to viral meningitis and/or viral encephalitis of unknown etiology. Three cases of seroreactivity to TBEV antigens were discovered among convalescent patients who recovered from viral meningitis and/or encephalitis and accepted to participate in the study (n = 15). The clinical and laboratory findings of these patients overlap with that of seronegative convalescent patients. Although TBE has been a notifiable disease in Serbia since 2004, there is no active TBE surveillance program for the serologic or molecular screening of TBEV infection in humans in the country. This study highlights the necessity to increase the awareness of TBE among physicians and perform active and systematic screening of TBEV antibodies among patients with viral meningitis and/or encephalitis.

A three-dimensional evaluation of microleakage of class V cavities prepared by the very short pulse mode of the erbium:yttrium-aluminium-garnet laser.

The aim of this study was to evaluate microleakage along resin restoration in cavities prepared with an erbium:yttrium-aluminium-garnet (Er:YAG) laser, with and without acid etching, and to compare it with that in diamond-drilled cavities. Thirty intact molars were divided into three equal groups. In the teeth in group I, class V cavities were prepared with a diamond drill. Cavities in groups II and III were prepared with an Er:YAG laser (400 mJ/15 Hz for enamel and 250 mJ/10 Hz for dentine). The cavities in groups I and II were acid-etched and adhesive and flowable composite were applied to all cavities. The specimens were first immersed in dye for 24 h and then in 5% nitric acid for 72 h for softening. The fillings were extracted and photographed through a dissecting microscope. The leakage area was measured with specially designed software. The Kruskal-Wallis test showed that the best ranking was group II [mean range (m.r.) = 27.46], followed by group I (m.r. = 33.48) and, lastly, group III (m.r. = 45.15). The differences between groups I and III (P = 0.023) and between groups II and III were statistically significant (P = 0.080). The least microleakage was found in those cavities prepared by Er:YAG laser and subsequently acid-etched, whereas the most leakage was in the lased cavities that had not been etched; the traditional diamond-drilled acid-etched cavities produced medium leakage.