Tick-borne diseases under the radar in the North Sea Region

The impact of tick-borne diseases caused by pathogens such as Anaplasma phagocytophilum, Neoehrlichia mikur-ensis, Borrelia miyamotoi, Rickettsia helvetica and Babesia species on public health is largely unknown. Data on the prevalence of these pathogens in Ixodes ricinus ticks from seven countries within the North Sea Region in Europe as well as the types and availability of diagnostic tests and the main clinical features of their corresponding diseases is reported and discussed. Raised awareness is needed to discover cases of these under-recognized types of tick-borne disease, which should provide valuable insights into these diseases


Background
Tick-borne diseases present a growing health concern for humans worldwide.Ixodes ricinus ticks are the main vectors of pathogens causing human tick-borne infections in Europe.The most familiar infectious agents include Borrelia burgdorferi sensu lato and tick-borne encephalitis virus, which may cause Lyme borreliosis and tick-borne encephalitis, respectively, for which European centre for Disease Prevention and Control guidelines are available (ECDC, 2023).I. ricinus may also transmit lesser-known pathogens, such as Anaplasma phagocytophilum, Neoehrlichia mikurensis, Borrelia miyamotoi, Rickettsia helvetica and Babesia species, all of which can cause human disease rarely diagnosed in Europe (Sprong et al., 2018).The impact of these pathogens on public health is unclear.Clinicians' lack of experience on how these infections manifest and limited availability of proper diagnostic services jointly contribute to their likely underdiagnosis.
The goal of this work is to raise awareness of the under-recognized tick-borne diseases caused by A. phagocytophilum (anaplasmosis), N. mikurensis (neoehrlichiosis), B. miyamotoi (B.miyamotoi disease), R. helvetica (R. helvetica infection) and Babesia spp.(babesiosis) and the laboratory testing methods currently available, thereby contributing to improved patient management and recognition of the varied manifestations of these illnesses.This work was performed as part of NorthTick, a project co-funded by the European Union through the European Regional Development Fund and the North Sea Region Program.The geographic study area encompassed seven countries (Belgium, Denmark, Germany, Netherlands, Norway, Scotland and Sweden) participating in the project (Fig. 1).

Pathogen prevalence and infection risk
The probability of contracting a tick-borne disease from a tick bite depends on several factors, such as the risk of being bitten by a tick carrying a pathogen, the ability of the pathogen to be transmitted to the human host and its potency to cause symptoms and manifest as a disease (Sprong et al., 2018).The risk of developing disease versus an asymptomatic seroconversion also depends on the immune status of the host.Individuals with an impaired immunity, due to either primary immunodeficiency or immunosuppressive disease or treatment, are at higher risk of developing more severe symptoms than immunocompetent individuals.
Knowledge on how many individuals in Europe that have acquired a certain tick-borne disease is crucial for understanding the risk of infection and its impact on public health, but the number of infected individuals is generally not known due to insufficient investigation of cases and the lack of national surveillance and notification systems.
The infection rates of pathogens in I. ricinus ticks within defined geographical regions may also provide useful information on the risk of contracting a tick-borne diseases from a tick bite.Table 1 summarizes the prevalence rates of the less common tick pathogens in I. ricinus reported in the scientific literature, providing an overview of the pathogen distribution in the North Sea Region countries.It should be noted that the data is limited and potentially skewed due to factors as use of different methodologies for detection, small sample sizes (few ticks studied), and the reservoir, i.e., host animals for the various tick-borne pathogens in the areas where the ticks were collected (Sprong et al., 2018).
The overall prevalence rates of A. phagocytophilum and B. miyamotoi in I. ricinus seem to be quite similar in all seven North Sea Region countries.The rate of A. phagocytophilum differs greatly from site to site but the majority of studies report around 1-5% infected ticks (Blazejak et al., 2017;Coipan et al., 2013;Flattery et al., 2022;Franke et al., 2010;  The range of the most commonly or all (when data is limited) reported pathogen prevalence rates in ticks are given, together with the spreading of all data (in parenthesis).Data was obtain by searching PubMed using combinations of the pathogen names and the country names together with Ixodes ricinus.Data on questing ticks was primarily included with a few exceptions (marked with *).*Only data from ticks collected from animals was available and presented (Abdullah et al., 2018;Lempereur et al., 2011).All other references are given in the main text.
H. Quarsten et al.Schorn et al., 2011;Silaghi et al., 2012b;Wielinga et al., 2009;Øines et al., 2012).Babesia divergens and Babesia venatorum are present in all countries with the latter species most frequently detected, whereas Babesia microti has not yet been detected in Denmark and Norway.However, there is no systematic tick-borne pathogen surveillance of ticks in the North Sea Region countries to date.N. mikurensis and/or R. helvetica are the two pathogens that appear to infect the highest number of I. ricinus in many regions and if their transmission from tick to human is effective, there could be a high risk of being exposed to the bacteria when tick-bitten.The ability of both organisms to cause disease, however, seems to be very different since the numbers of diagnosed disease cases caused by N. mikurensis have increased successively since the first case reports were published in 2010, whereas published cases attributed to R. helvetica are scarce (Höper et al., 2020;Nilsson, 2009Nilsson, , 1999bNilsson, , 2010Nilsson, , 2011;;Wenneras, 2015).

Laboratory diagnosis of the under-recognized tick-borne diseases
Laboratory diagnosis of the under-recognized tick-borne diseases is a challenge.None of the pathogens involved is detected by the routine culture methods available in diagnostic microbiology laboratories.A. phagocytophilum, N. mikurensis and R. helvetica are intracellular bacteria that depend on cell lines for culture, and B. miyamotoi and the intraerythrocytic protozoan parasite Babesia are both fastidious and very difficult to culture without particular expertise.The three main categories of diagnostic methodologies are molecular (detection of pathogen DNA), serologic (detection of antibodies against the pathogen) or morphologic (microscopic examination of the patient's blood) analysis.The most common methods used for diagnosing the tick-borne diseases are listed in Table 2.
Direct detection of pathogen DNA by specific PCR or 16S (prokaryotic)/18S (eukaryotic)-based amplification and sequencing is generally useful in the early stage of the illness when higher amounts of the pathogen are present.PCR methods are established for all five pathogens, and they may be either species-or genus/group-(as for spotted fever group Rickettsia) specific.Commercially available kits exist for A. phagocytophilum, B. miyamotoi, Rickettsia spp.and Babesia spp., although many diagnostic laboratories use laboratory developed ("inhouse") assays.
The 16S/18S-sequencing approach may be particularly advantageous in cases where the etiology is uncertain and there is no explicit suspicion of tick-borne diseases since the methods essentially will detect all types of prokaryotic/eukaryotic DNA.However, sensitivity is lower than for specific PCR. A. phagocytophilum (infecting neutrophilic granulocytes), Babesia (infecting red blood cells) together with N. mikurensis (infecting the endothelium lining the blood vessels) and B. miyamotoi (existing extracellularly) are pathogens normally detected in blood (Bakken et al., 1994;Kawahara et al., 2004;Krause et al., 2015;Rudzinska et al., 1976;Wass et al., 2019), whereas it is unknown if R. helvetica is present in blood in the acute phase of disease.
Molecular testing of whole blood is the method of choice for all symptomatic under-recognized tick-borne diseases except for the rickettsioses.Most rickettsioses diagnosed in the North Sea Region are infections imported from the southern part of Europe, such as the Mediterranean spotted fever caused by R. conorii, or elsewhere in the world, such as the African tick-bite fever caused by R. africae (Oteo and Portillo, 2012).The amount of rickettsial DNA in blood is generally low and the sensitivity of molecular testing in blood is suboptimal (Stewart and Stewart, 2021).A possible pitfall when diagnosing B. miyamotoi disease may be the relapsing nature of the disease, as B. miyamotoi is detectable for only around four days during a febrile episode (Karan et al., 2018).Thus, B. miyamotoi DNA may no longer be detectable in blood at the time the patient presents with an infection of the central nervous system (Boden et al., 2016).B. miyamotoi and rickettsial DNA can be detected in cerebrospinal fluid of patients with neurological symptoms, so molecular testing of cerebrospinal fluid should be considered (Hoornstra et al., 2022;Nilsson et al., 2010Nilsson et al., , 2011)).
Serological assays for the detection of either species-or group- Long-term persistence of antibodies from a previous pathogen exposure or infection may be mistaken as a current infection.In areas, highly endemic for tick-borne disease, seroprevalence rates against common tick-borne pathogens may be high, making it challenging to interpret serologic findings.Cross-reactive antibodies, especially of IgM class, may also be misleading.Antibodies produced against N. mikurensis are reported to cross-react in A. phagocytophilum antibody tests in such a way that a patient with neoehrlichiosis may be misdiagnosed as having anaplasmosis (Wass et al., 2018).Furthermore, B. miyamotoi infection may give rise to antibodies in cerebrospinal fluid that cross-react in B. burgdorferi C6 antibody tests (Koetsveld et al., 2020), a result that may be misinterpreted as Lyme neuroborreliosis (Hoornstra et al., 2018;Sudhindra et al., 2016).Another possible pitfall in serology with a particular impact on the under-recognized tick-borne diseases is that immunosuppressed individuals may have false negative tests due to impaired ability to produce antibodies when infected (Henningsson et al., 2019;Tavakolpour et al., 2019).Altogether, this underlines why molecular testing is the preferred diagnostic method and of particular importance when testing immunosuppressed patients for the under-recognized tick-borne diseases.In spite of that, antibody testing may be useful for the diagnosis of low-grade infections, such as between relapses of B. miyamotoi disease.The development of a serological method for neoehrlichiosis is also anticipated, which may enable the detection of the low-grade persistent type of infection observed among N. mikurensis exposed individuals (Grankvist et al., 2015;Quarsten et al., 2021;Welc-Faleciak et al., 2014).In addition, serology remains an important tool for seroprevalence surveys that may provide crucial information on the rate of pathogen exposure in a population.Microscopy of Giemsa-stained blood smears is a well-established method for the identification of Babesia-infected red blood cells in endemic areas, whereas microscopic examination of stained acute phase blood smears for detection of intracytoplasmic morulae in anaplasmosis patients is less useful due to low diagnostic sensitivity for European A. phagocytophilum variants (Lotric-Furlan et al., 2006).Microscopic detection of Babesia in blood cells is technically easy to perform and may be done at local hospital laboratories, but it may be mistaken for Plasmodium falciparum malaria by inexperienced operators (Kukina et al., 2018).In practice, due to low awareness only a few specialized laboratories are experienced and use the technique.

Clinical features of the under-recognized tick-borne diseases
To understand when to suspect and test for one of the less established tick-borne diseases is demanding.Clinicians should remember to ask patients with unexplained illnesses about their history of tick bites.However, it is important to keep in mind that a previous tick bite may not always have been recognized by the patient since a large fraction of them pass unnoticed.
Most of the infections caused by the under-recognized tick-borne pathogens in immunocompetent individuals are probably asymptomatic or mild and self-limiting and do not require treatment.However, symptomatic cases occur and often but not always, manifest as a nonspecific febrile illness with myalgia, and may warrant antibiotic treatment.In immunosuppressed patients, the symptoms can be more severe and even life-threatening making it crucial to identify such patients.Recognizing the clinical manifestations and biochemical laboratory findings indicating the lesser-known tick-borne diseases may, however, be challenging.Symptoms and disease features important for clinicians to be aware of are summarized in a diagnostic flowchart (Fig. 2) and briefly described.A more detailed description of the clinical and laboratory parameters associated with the various tick-borne diseases are beyond the scope of this work and should be pursued elsewhere.
Fever is a dominating symptom of most under-recognized tick-borne diseases.Immunosuppressed neoehrlichiosis patients often have episodes with daily fever (Wenneras, 2015).Patients with B. miyamotoi disease may have intermittent fever interspersed with afebrile episodes (recurrent fever) often totaling two or three relapse periods (Platonov et al., 2011;Wagemakers et al., 2015).Babesiosis may also present with relapsing fever episodes (Shuker et al., 2018).Febrile hemolytic disease, a clinical manifestation mimicking malaria, is an indication of babesiosis.Malaria, acquired during travels to endemic areas, is more common and should always be excluded as the cause of infection before testing for Babesia.
Increased liver enzymes are acknowledged to be common in anaplasmosis and babesiosis cases and could indicate testing for those particular infections, however, a certain fraction of all the underrecognized tick-borne disease patients will also express the same abnormality (Azagi et al., 2020;Dumic et al., 2022).A discriminatory finding that may be useful is blood platelet levels, which tend to be decreased in anaplasmosis and normal or even increased in neoehrlichiosis (Grankvist et al., 2014;Matei et al., 2019).Further, moderately increased white blood cells dominated by neutrophils is also a common feature of neoehrlichiosis (Wenneras, 2015).
Acute disease caused by N. mikurensis, B. miyamotoi or Babesia is most often, but not exclusively, diagnosed in immunocompromised patients.Rituximab (anti-CD20/B cell) treatment is a predominant risk factor for severe neoehrlichiosis and B. miyamotoi disease, whereas the majority of patients diagnosed with severe babesiosis together with a fraction of the neoehrlichiosis patients are asplenic (Hildebrandt et al., 2021;Wagemakers et al., 2015;Wenneras, 2015).On the contrary, many European anaplasmosis cases are diagnosed in immunocompetent individuals, although with milder manifestations than seen in the immunosuppressed (Azagi et al., 2020).The few reported cases of severe infection caused by R. helvetica are from individuals with intact immunity (Nilsson, 2009(Nilsson, , 1999b(Nilsson, , 2010(Nilsson, , 2011)).
Infected immunocompromised patients may often have a prolonged (for months) disease course as seen in several neoehrlichiosis and babesiosis patients as well as in some of the few reported cases of B. miyamotoi-meningitis (Henningsson et al., 2019;Wagemakers et al., 2015;Wenneras, 2015).Infections may be mistaken for non-infectious conditions in immunocompromised patients with underlying medical conditions, even when the burden of symptoms is severe, because routine microbiologic investigations remain negative (Grankvist et al., 2014).A hallmark of severe neoehrlichiosis, independent of immune status of the patient, is manifestation of vascular complications (Wenneras, 2015).Thromboembolic events in the venous circulation (thrombophlebitis, deep vein thrombosis, pulmonary embolism) seem to affect immunocompromised individuals whereas inflammation of medium-to large-sized arteries have only been seen in immunocompetent patients infected by N. mikurensis (Höper et al., 2020).Neoehrlichiosis is often diagnosed at the time the patient presents with atypical vascular and/or thromboembolic events with no obvious risk factors.More focus on testing for neoehrlichiosis in patients (in particular Rituximab treated) with fever and vascular events may prevent further potentially life-threatening complications that can be eradicated by antibiotic treatment (Sjöwall et al., 2021).
Neurological infection is solely a complication of severe disease caused by B. miyamotoi or R. helvetica, yet critical cases seem to be rare for both types of infections (Azagi et al., 2020;Cutler et al., 2019).Recurrent episodes of neurological symptoms with symptom-free intervals, may indicate relapsing B. miyamotoi meningitis (Henningsson et al., 2019).
The under-recognized tick-borne diseases are not treated with the first-choice antibiotics commonly used for infections of unknown origin.Therefore, diagnosing the tick-borne diseases is of major importance for correct treatment and management of patients.Infections caused by the intracellular bacteria A. phagocytophilum, N. mikurensis and R. helvetica are primarily treated by high-dose doxycycline (Wass et al., 2018).Doxycycline is also the drug of choice for B. miyamotoi disease, including B. miyamotoi meningitis (Henningsson et al., 2019).Cases of babesiosis require treatment with antiparasitic drugs, e.g.atovaquone or quinine, given in combination with azithromycin or clindamycin.A missed diagnosis may delay or hinder correct treatment, putting the patient at risk of an extended infection period and severe complications.

Availability of diagnostic services for the under-recognized tick-borne diseases
As part of the work of the NorthTick project, a brief survey on the availability of diagnostic services within the countries in the North Sea Region was conducted.It was demanding to get a reliable overview of all seven countries.Methods for the detection of the under-recognized tickborne diseases validated for clinical use are available in only a few laboratories.Most countries seemed to have at least one laboratory offering serological and/or molecular diagnostic services for the tickborne diseases caused by A. phagocytophilum, B. miyamotoi, Rickettsia and Babesia.The lack of an established laboratory method for detection of babesiosis in some countries may to a certain degree be compensated by using microscopic examination of blood in hospital laboratories.Some countries also have established international collaborations to fill in the methodological gaps.The most evident finding from the assessment was that the Scandinavian countries provide routine molecular testing for N. mikurensis, reflecting a higher awareness of neoehrlichiosis and a higher number of cases in those countries than in the non-Scandinavian countries of the North Sea Region.

Considerations on the diagnostic services of the underrecognized tick-borne diseases
As discussed earlier, A. phagocytophilum, N. mikurensis, B. miyamotoi and Babesia are carried by ticks in most North Sea Region countries, even if not always widespread.Although they apparently do not have a high potential to cause severe infections in immunocompetent individuals, evidence indicating a significant impact on infected immunocompromised individuals is growing (Hildebrandt et al., 2021;Hoornstra et al., 2022;Wenneras, 2015).The number of individuals at risk of contracting a severe infection and complications is continuously increasing due to the rising use of biological therapy for many common autoimmune and inflammatory diseases such as rheumatoid arthritis, multiple sclerosis and inflammatory bowel disease, as well as for malignant B cell lymphomas.
The diagnostic services available should be harmonized with the anticipated need.Establishing a broad test repertoire in several laboratories in each country may be needless and costly.The authors suggest that the countries within the North Sea Region could consider consolidating diagnostic services to one or a few national reference centers.This would promote an increased level of competence and expertise at each center, facilitate research and make referral pathways clearer for testing and clinical advice.Closing significant diagnostic gaps in the national test repertoire could also be done by taking advantage of the diagnostics available in national reference or specialized laboratories in partner countries.This testing should be advertised and available for the clinicians upon request in the same manner as the nationally provided tests.Since there are no national surveillance systems for the more uncommon tick-borne diseases, a collected diagnostics competence will facilitate a better overview of diagnosed cases.However, national surveillance and notification services would be ideal especially since tickborne diseases are anticipated to increase in the wake of climate change.
The national diagnostic services should primarily be molecularbased but should also include antibody testing and microscopy where appropriate and available.The symptoms of the under-recognized tickborne diseases often resemble each other to a high degree, often featuring fever and myalgia, and clinicians rarely have experience on how to distinguish between these infections.Thus, choosing diagnostic strategies like multiplex PCR (or panel testing) to detect all relevant tickborne pathogens will help increase the number of correctly diagnosed cases and contribute to improved knowledge.Clinicians suspecting an under-recognized tick-borne disease regularly request A. phagocytophilum testing only since anaplasmosis is the infection most familiar to them.However, in many regions, as seen in the southern part of Norway and Sweden, it is much more likely to contract neoehrlichiosis.
The need for specific tests for detection of R. helvetica is less clear as only a few severe cases of disease are known and a certain causality between tick exposure and disease is not established (Azagi et al., 2020).However, several countries are providing tests for detection of the rickettsial infections occasionally imported from the southern parts of Europe or wider afield.These methods may be valuable for identification of infection caused by R. helvetica given that serological assays detecting particular Rickettsia spp.are frequently cross-reactive for other spotted fever group Rickettsia and molecular assays often detect a broad range of Rickettsia spp., including R. helvetica.

Closing remarks
The majority of infections caused by the under-recognized tick-borne diseases are thought to be asymptomatic or mild and self-limiting.However, their ability to cause severe disease, especially in the immunocompromised population, requires serious consideration.Due to the lack of characteristic symptoms, low awareness, absence of surveillance and limited use/availability of diagnostic tests for the under-recognized tick-borne diseases, most cases probably remain undiagnosed and the impact on public health remains unclear.Focus on raising the awareness of the infections among clinicians and clinical microbiologists and proper organization and evaluation of the diagnostic services is of high importance, and will together facilitate an increase in targeted testing and detection of more disease cases.Each new diagnosed tick-borne disease case will provide valuable epidemiological data on incidence and prevalence as well as increasing of important understanding of clinical manifestations.Rapid and accurate diagnosis is crucial to ensure adequate antimicrobial therapy when needed and reduce the number of patients with complications.

Fig. 1 .
Fig. 1.An overview of the countries within the North Sea Region, participating in the NorthTick project.Interreg North Sea Region gave the permission to use the map.

Fig. 2 .
Fig. 2. Diagnostic flowchart for the under-recognized tick-borne diseases in the North Sea Region.

Table 1
Reported pathogen prevalence in Ixodes ricinus ticks within the investigated North Sea Region countries.

Table 2
Methods currently available for diagnosing the under-recognized human tick-borne diseases.antibodies are established for all under-recognized tick-borne pathogens with the exception of N. mikurensis.Assays are generally commercially available, but only a few specialized clinical laboratories in Europe have tests validated for clinical use.Specific antibodies often take weeks to develop in response to infection, so serological tests may not be useful in the early phase of disease.A four-fold change in IgG titer in paired serum samples taken 2-4 weeks apart confirms a diagnosis.Furthermore, the presence of IgM may support the diagnosis, even after a single test result.A single positive antibody test should be interpreted carefully.
H.Quarsten et al.specific