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•In 2022, the study enrolled 819 patients from Serbia and North Macedonia.•Subclinical bacteremia occurred in 8.06% of cases, primarily with Anaplasmaspp.•Uncharacterized Rickettsiaspp. and SFGR were dominant in North Macedonia and Serbia.•Phylogenetic analysis revealed high genetic diversity among the identified pathogens.

Tick-borne pathogens (TBPs) pose a major threat to human health in Europe and the whole northern hemisphere. Despite a high prevalence of TBPs in Ixodes ricinusticks, knowledge on the incidence of tick-borne diseases in humans infested by this tick species is limited. This study was conducted in the year 2019 on patients who presented themselves to the Pasteur Institute Novi Sad with tick infestations. Ticks (n= 31) feeding on human (n= 30) and blood samples from the same individuals were collected by physicians and a microfluidic real-time high-throughput PCR system was used to test the genomic DNA of the samples for the presence of 27 bacterial and eight parasitic microorganisms in Serbia. Except for one Rhipicephalus sanguineuss.l. adult male tick, all ticks infesting humans were morphologically identified as I. ricinus. A high proportion of ticks (74 %, 23/31) were infected with at least one of the tested TB microorganisms, being Rickettsia helvetica(54 %, 17/31) the most common pathogen, but Borrelia afzelii(9 %, 3/31), Anaplasma phagocytophilum(6 %, 2/31), Borrelia miyamotoi(6 %, 2/31), and Francisellalike-endosymbiont (6 %, 2/31), Borrelia valaisiana(3 %, 1/31), Borrelia lusitaniae(3 %, 1/31), Rickettsia felis(3 %, 1/31) and Rickettsia aeschlimannii(3 %, 1/31) were also identified. Despite the high infection rate of TBPs in ticks, only two human blood samples (6 %, 2/30) tested positive for the presence of TBPs, one patient (code H12, 67 years old female) was diagnosed with Borreliaspp. and the other patient was diagnosed (code H17, 71 years old female) with R. felisinfection. The tick infesting patient H12 tested positive for B. afzelii, and R. helveticaand the tick infesting patient H17 tested positive for R. felis. Upon clinical examination, both patients were diagnosed with erythema migrans. No additional discomfort was reported by the patient and no additional pathology was observed by the physician. We concluded that humans bitten by I. ricinusin Serbia are exposed to a diverse array of TBPs with clinical impact in the Serbian cohort studied.