Molecular Detection of Tick-Borne Pathogens in Humans with Tick Bites and Erythema Migrans, in the Netherlands

Abstract

Background: Tick-borne diseases are the most prevalent vector-borne diseases in Europe. Knowledge on the incidence and clinical presentation of other tick-borne diseases than Lyme borreliosis and tick-borne encephalitis is minimal, despite the high human exposure to these pathogens through tick bites. Using molecular detection techniques, the frequency of tick-borne infections after exposure through tick bites was estimated.

Methods: Ticks, blood samples and questionnaires on health status were collected from patients that visited their general practitioner with a tick bite or erythema migrans in 2007 and 2008. The presence of several tick-borne pathogens in 314 ticks and 626 blood samples of this cohort were analyzed using PCR-based methods. Using multivariate logistic regression, associations were explored between pathogens detected in blood and self-reported symptoms at enrolment and during a three-month follow-up period.

Results: Half of the ticks removed from humans tested positive for Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, Candidatus Neoehrlichia mikurensis, Rickettsia helvetica, Rickettsia monacensis, Borrelia miyamotoi and several Babesia species. Among 92 Borrelia burgdorferi s. l. positive ticks, 33% carried another pathogen from a different genus. In blood of sixteen out of 626 persons with tick bites or erythema migrans, DNA was detected from Candidatus Neoehrlichia mikurensis (n = 7), Anaplasma phagocytophilum (n = 5), Babesia divergens (n = 3), Borrelia miyamotoi (n = 1) and Borrelia burgdorferi s. l. (n = 1). None of these sixteen individuals reported any overt symptoms that would indicate a corresponding illness during the three-month follow-up period. No associations were found between the presence of pathogen DNA in blood and; self-reported symptoms, with pathogen DNA in the corresponding ticks (n = 8), reported tick attachment duration, tick engorgement, or antibiotic treatment at enrolment.

Conclusions: Based on molecular detection techniques, the probability of infection with a tick-borne pathogen other than Lyme spirochetes after a tick bite is roughly 2.4%, in the Netherlands. Similarly, among patients with erythema migrans, the probability of a co-infection with another tick-borne pathogen is approximately 2.7%. How often these infections cause disease symptoms or to what extend co-infections affect the course of Lyme borreliosis needs further investigations.

Fig 1. Phylogenetic tree of the sequences obtained from human blood samples.

PCR and sequencing was performed on the real-time PCR-positive blood samples. Sequences were clustered with (reference) sequences from Genbank. The evolutionary distance values were determined by Kimura method, and the tree was constructed according to the neighbor-joining method. A.) Anaplasma phagocytophilum: Phylogenetic tree of partial heat shock protein gene groEL of Anaplasma phagocytophilum of the four, one sequences is slightly different by couple of mismatches. All four are part of zoonotic variant of Anaplasma phagocytophilum. B.) Babesia genospecies: Three of the tested blood samples for Babesia genospecies yielded a sequence for the ribosomal 18S rRNA gene, and showed to be identical to B. divergens sequences. C.) Candidatus Neoehrlichia mikurensis: Five out of seven Candidatus Neoehrlichia mikurensis yielded a partial sequence of the citrate synthase gene gltA. D.) Candidatus Neoehrlichia mikurensis: All seven Candidatus Neoehrlichia mikurensis yielded a partial sequence of the heat shock protein gene groEL.