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Background A number of tick-borne pathogens circulate in the Belgian tick population in addition to the causative agent of Lyme borreliosis. However, so far, only a few patients with tick-borne diseases other than Lyme borreliosis have been reported in Belgium. The aim of this study was to investigate the occurrence of other human tick-borne infections in Belgium and their possible clinical manifestation. Methods Patients with fever (&gt; 37.5 °C) after a tick bite or those with erythema migrans (EM) were included in the study. EDTA-blood samples were screened for the presence of DNA from  Borrelia burgdorferi  sensu lato,  Borrelia miyamotoi ,  Anaplasma phagocytophilum ,  Neoehrlichia mikurensis , spotted fever group rickettsiae (genus  Rickettsia ),  Babesia  spp.,  Bartonella  spp.,  Spiroplasma ixodetis  and tick-borne encephalitis virus, using multiplex PCR methods. A questionnaire on, among others, demographics and clinical symptoms, was also filled in. Results Over a period of 3 years, 119 patients with EM and 14 patients with fever after a recent tick bite were enrolled in the study. Three samples initially tested positive for  N. mikurensis  by quantitative PCR (qPCR), but the results could not be confirmed by other PCR methods, and repetition of the DNA extraction procedure and qPCR test was not successful. The qPCR test results for the other tick-borne pathogens were negative. Conclusions In general, only a few patients with fever after a tick bite could be identified. Although no tick-borne pathogens were detected, their occurrence cannot be excluded based on the limited number of patients and the limitations inherent to current methodologies. This study underscores the possibility of false-positive PCR results and the necessity for the development of multiple independent tools for the sensitive and specific detection of emerging tick-borne pathogens. Graphical Abstract

No molecular detection of tick-borne pathogens in the blood of patients with erythema migrans in Belgium