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1.  Molecular Detection of Tick-Borne Pathogens in Humans with Tick Bites and Erythema Migrans, in the Netherlands 
PLoS Neglected Tropical Diseases  2016;10(10):e0005042.
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.
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.
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.
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.
Author Summary
Two most common tick-borne diseases in Europe are Lyme borreliosis and tick-borne encephalitis. Ticks transmit many more pathogens, causing neglected diseases such as anaplasmosis, babesiosis, rickettsiosis and neoehrlichiosis. These diseases are seldom diagnosed, due to their mild and non-characteristic symptoms, but also due to lack of awareness and availability of diagnostic tests. Using molecular detection techniques (polymerase chain reaction or PCR), we estimated the frequency of tick-borne infections in humans after a tick bite and in patients with the first symptoms of Lyme borreliosis, an erythema migrans. About half of the ticks that fed on humans carried one or more tick-borne pathogens, and approximately 2.5% of people that were bitten by ticks were infected with a tick-borne pathogen other than Lyme borreliosis or tick-borne encephalitis. Co-infections of a tick-borne pathogen in patients with an erythema migrans was also approximately 2.5%. Based on these findings, we estimated the incidence of tick-borne infections other than Lyme borreliosis in the Netherlands. How often these infections cause disease or to what extend co-infections affect the course of Lyme borreliosis needs further investigations.
PMCID: PMC5051699  PMID: 27706159
2.  Decrease in tick bite consultations and stabilization of early Lyme borreliosis in the Netherlands in 2014 after 15 years of continuous increase 
BMC Public Health  2016;16:425.
Nationwide surveys have shown a threefold increase in general practitioner (GP) consultations for tick bites and early Lyme borreliosis from 1994 to 2009 in the Netherlands. We now report an update on 2014, with identical methods as for the preceding GP surveys.
To all GPs in the Netherlands, a postal questionnaire was sent inquiring about the number of consultations for tick bites and erythema migrans diagnoses (most common manifestation of early Lyme borreliosis) in 2014, and the size of their practice populations.
Contrasting to the previously rising incidence of consultations for tick bites between 1994 and 2009, the incidence decreased in 2014 to 488 consultations for tick bites per 100,000 inhabitants, i.e., 82,000 patients nationwide. This survey revealed a first sign of stabilization of the previously rising trend in GP diagnosed erythema migrans, with 140 diagnoses per 100,000 inhabitants of the Netherlands. This equals about 23,500 annual diagnoses of erythema migrans nationwide in 2014.
In contrast to the constantly rising incidence of GP consultations for tick bites and erythema migrans diagnoses in the Netherlands between 1994 and 2009, the current survey of 2014 showed a first sign of stabilization of erythema migrans diagnoses and a decreased incidence for tick bite consultations.
PMCID: PMC4877959  PMID: 27216719
Tick bites; Erythema migrans; Early Lyme borreliosis; Incidence
4.  Physician reported incidence of early and late Lyme borreliosis 
Parasites & Vectors  2015;8:161.
Lyme borreliosis is the most common vector-borne disease in Europe and North America. The objective of this study is to estimate the incidence of tick bites and Lyme borreliosis, representative of our entire country, including erythema migrans, disseminated Lyme borreliosis and persisting symptoms attributed to Lyme borreliosis.
A questionnaire on clinical diagnoses of Lyme borreliosis was sent to all GPs, company physicians, and medical specialists. To adjust for possible misclassification and telescoping bias, we sent additional questionnaires to categorize reported cases according to likelihood of the diagnosis and to exclude cases diagnosed outside the target period.
Adjusted annual incidence rate for disseminated Lyme borreliosis was 7.7 GP reports per 100,000 inhabitants, and for persisting symptoms attributed to Lyme borreliosis was 5.5 GP reports per 100,000 inhabitants, i.e. approximately 1,300 and 900 cases respectively. GP consultations for tick bites and erythema migrans diagnoses were 495 and 132 per 100,000 inhabitants, respectively, i.e. 82,000 and 22,000 cases in 2010.
This is the first reported nationwide physician survey on the incidence of tick bites and the whole range of manifestations of Lyme borreliosis, including persisting symptoms attributed to Lyme borreliosis. This is crucial for complete assessment of the public health impact of Lyme borreliosis.
Electronic supplementary material
The online version of this article (doi:10.1186/s13071-015-0777-6) contains supplementary material, which is available to authorized users.
PMCID: PMC4363353  PMID: 25889086
Lyme borreliosis; Borrelia burgdorferi; Incidence; Medical diagnoses
5.  Human Exposure to Tickborne Relapsing Fever Spirochete Borrelia miyamotoi, the Netherlands 
Emerging Infectious Diseases  2014;20(7):1244-1246.
PMCID: PMC4073841  PMID: 24963562
Tickborne diseases; zoonoses; relapsing fever; Lyme disease; Borrelia; borreliosis; miyamotoi; B. burgdorferi sensu lato; the Netherlands
6.  A Prospective Study among Patients Presenting at the General Practitioner with a Tick Bite or Erythema Migrans in the Netherlands 
PLoS ONE  2013;8(5):e64361.
We performed a nationwide prospective study on the transmission risk for Borrelia to humans, investigating symptoms and serology at enrolment and three months after tick bites, and after standard treatment for erythema migrans (EM). Aiming to quantify the infection risk at point of care by physicians, we explored risk factors such as tick testing for Borrelia and assessment of the duration of the tick's blood meal.
Methods and Findings
Questionnaires, blood samples and ticks from patients who consulted one of 307 general practitioners for tick bites (n = 327) or EM (n = 283) in 2007 and 2008, were collected at enrolment and three months later at follow-up. Borrelia burgdorferi sensu lato DNA was detected in 29.3% of 314 ticks, using PCR/reverse line blot and real-time PCR on the OspA gene. Seroconversion in C6 ELISA, IgM or IgG immunoblots for Borrelia-specific antibodies was observed in 3.2% of tick bite cases. Fourteen tick bite cases had evidence of early Borrelia infection, of which EM developed among seven cases. The risk of developing EM after tick bites was 2.6% (95%CI: 1.1%–5.0%), and the risk of either EM or seroconversion was 5.1% (95%CI: 2.9%–8.2%). Participants with Borrelia-positive ticks had a significantly higher risk of either EM or seroconversion (odds ratio 4.8, 95%CI: 1.1–20.4), and of seroconversion alone (odds ratio 11.1, 95%CI: 1.1–108.9). A third (34%) of the cases enrolled with EM did not recall preceding tick bites. Three EM cases (1%) reported persisting symptoms, three months after standard antibiotic treatment for EM.
One out of forty participants developed EM within three months after tick bites. The infection risk can be assessed by tick testing for Borrelia at point of care by physicians. However, further refining is needed considering sensitivity and specificity of tick tests, accuracy of tick attachment time and engorgement.
PMCID: PMC3655959  PMID: 23696884
7.  Circumstantial evidence for an increase in the total number and activity of borrelia-infected ixodes ricinus in the Netherlands 
Parasites & Vectors  2012;5:294.
Between 1994 and 2009, a threefold increase has been observed in consultations of general practitioners for tick bites and Lyme disease in The Netherlands. The objective of this study was to determine whether an increase in the number of questing ticks infected with B. burgdorferi sensu lato is a potential cause of the rise in Lyme disease incidence.
Historic data on land usage, temperature and wildlife populations were collected and analyzed together with data from two longitudinal field studies on density of questing ticks. Effective population sizes of Borrelia burgdorferi s.l. were calculated.
Long-term trend analyses indicated that the length of the annual tick questing season increased as well as the surface area of tick-suitable habitats in The Netherlands. The overall abundances of feeding and reproductive hosts also increased. Mathematical analysis of the data from the field studies demonstrated an increase in mean densities/activities of questing ticks, particularly of larvae between 2006 and 2009. No increase in infection rate of ticks with Borrelia burgdorferi sensu lato was found. Population genetic analysis of the collected Borrelia species points to an increase in B. afzelii and B. garinii populations.
Together, these findings indicate an increase in the total number of Borrelia-infected ticks, providing circumstantial evidence for an increase in the risk of acquiring a bite of a tick infected with B. burgdorferi s.l. Due to the high spatiotemporal variation of tick densities/activities, long-term longitudinal studies on population dynamics of I. ricinus are necessary to observe significant trends.
PMCID: PMC3562265  PMID: 23244453
Borrelia burgdorferi sensu lato; Ixodes ricinus; Population dynamics; Lyme disease; The Netherlands
8.  Small risk of developing symptomatic tick-borne diseases following a tick bite in the Netherlands 
Parasites & Vectors  2011;4:17.
In The Netherlands, the incidence of Lyme borreliosis is on the rise. Besides its causative agent, Borrelia burgdorferi s.l., other potential pathogens like Rickettsia, Babesia and Ehrlichia species are present in Ixodes ricinus ticks. The risk of disease associated with these microorganisms after tick-bites remains, however, largely unclear. A prospective study was performed to investigate how many persons with tick-bites develop localized or systemic symptoms and whether these are associated with tick-borne microorganisms.
In total, 297 Ixodes ricinus ticks were collected from 246 study participants who consulted a general practitioner on the island of Ameland for tick bites. Ticks were subjected to PCR to detect DNA of Borrelia burgdorferi s.l., Rickettsia spp., Babesia spp. or Ehrlichia/Anaplasma spp.. Sixteen percent of the collected ticks were positive for Borrelia burgdorferi s.l., 19% for Rickettsia spp., 12% for Ehrlichia/Anaplasma spp. and 10% for Babesia spp.. At least six months after the tick bite, study participants were interviewed on symptoms by means of a standard questionnaire. 14 out of 193 participants (8.3%) reported reddening at the bite site and 6 participants (4.1%) reported systemic symptoms. No association between symptoms and tick-borne microorganisms was found. Attachment duration ≥24 h was positively associated with reddening at the bite site and systemic symptoms. Using logistic regression techniques, reddening was positively correlated with presence of Borrelia afzelii, and having 'any symptoms' was positively associated with attachment duration.
The risk of contracting acute Lyme borreliosis, rickettsiosis, babesiosis or ehrlichiosis from a single tick bite was <1% in this study population.
PMCID: PMC3050846  PMID: 21310036

Results 1-8 (8)