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

Abstract

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 (> 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

Assembly and Comparison of Ca. Neoehrlichia mikurensis Genomes

“…One of the major obstacles to investigating how often and under what conditions Ca. N. mikurensis causes an infectious disease in humans, is its unequivocal detection in larger cohorts of patients with noncharacteristic disease symptoms [11,12] or in persons with a (recent) tick bite [13][14][15]. In other words, supportive laboratory diagnostics to detect and identify Ca.…”

Technical Evaluation of qPCR Multiplex Assays for the Detection of Ixodes ricinus-Borne Pathogens

“…However, due to the scarcity of confirmed patient material, such as blood samples and even DNA extracts of these rare TBD cases, the repeatability of the other targets could not be evaluated [32]. In addition, the occurrence of non-reproducible results [14] and the contamination of positive controls [32] should not be overlooked. Therefore, confirmation of positive test results for these rare diseases by independent (European) laboratories or by sequencing the PCR products is essential in making a reliable diagnosis, at least for the microorganisms that are not yet established as human pathogens or that do not occur regularly (e.g., Spiroplasma and Neoehrlichia).…”

“…Of important notice is that the detection of a microorganism is indicative of infection but not of disease causation and should be complemented by additional diagnostic modalities such as serology and, whenever possible, live culture [14]. An assay that could be of great added value next to the multiplex qPCRs is a multi-pathogen serological diagnostic assay for the combined antigen and antibody detection for multiple TBPs.…”

“…TBEV is the most frequent arboviral disease in Europe, with 2000-3500 human cases each year. Other tick-borne diseases (TBDs) are far less frequently reported in the general population [9,14,15]. Several studies have shown that there is substantial exposure of humans to these TBPs through tick bites, such as B. miyamotoi, A. phagocytophilum, Babesia spp., B. microti, N. mikurensis, R. helvetica and S. ixodetis [16][17][18].…”

Contemporary diagnostics for medically relevant fastidious microorganisms belonging to the genera Anaplasma,Bartonella,Coxiella,OrientiaandRickettsia

“…This implies that MALDI-ToF MS is the method of choice for bacterial identification where >10 4 –10 5 cells are available for testing. In many cases, and this accounts not only for infections caused by fastidious organisms but for all human pathogens, even the best and most appropriate clinical specimens contain a very small number of pathogens (Geebelen et al 2022 ). This might result in false-negative diagnostic tests and this should always be considered in the interpretation of test results and the design of a therapeutic approach.…”