Evaluation of Disease Causality of Rare Ixodes ricinus-Borne Infections in Europe

In Europe, Ixodes ricinus ticks transmit pathogens such as Borrelia burgdorferi sensu lato and tick-borne encephalitis virus (TBEV). In addition, there is evidence for transmission to humans from I. ricinus of Anaplasma phagocytophilum, Babesia divergens, Babesia microti, Babesia venatorum, Borrelia miyamotoi, Neoehrlichia mikurensis, Rickettsia helvetica and Rickettsia monacensis. However, whether infection with these potential tick-borne pathogens results in human disease has not been fully demonstrated for all of these tick-borne microorganisms. To evaluate the available evidence for a causative relation between infection and disease, the current study analyses European case reports published from 2008 to 2018, supplemented with information derived from epidemiological and experimental studies. The evidence for human disease causality in Europe found in this review appeared to be strongest for A. phagocytophilum and B. divergens. Nonetheless, some knowledge gaps still exist. Importantly, comprehensive evidence for pathogenicity is lacking for the remaining tick-borne microorganisms. Such evidence could be gathered best through prospective studies, for example, studies enrolling patients with a fever after a tick bite, the development of specific new serological tools, isolation of these microorganisms from ticks and patients and propagation in vitro, and through experimental studies.


Introduction
Lyme borreliosis and tick-borne encephalitis (TBE) are caused by infections with Borrelia burgdorferi sensu lato and tick-borne encephalitis virus (TBEV), respectively. These well-established tick-borne diseases are frequently reported in Europe, and several studies have indicated a rise in the incidence of these diseases over the last decades [1][2][3]. In Europe, both pathogens are transmitted to humans by the bite of Ixodes ricinus. During such a bite, humans might also be exposed and sometimes infected with other microorganisms transmitted by I. ricinus [4]. These potential tick-borne pathogens (TBPs) include Anaplasma phagocytophilum, Babesia divergens, Babesia microti, Babesia venatorum, Borrelia miyamotoi, Neoehrlichia mikurensis, Rickettsia helvetica, and Rickettsia monacensis, which have all been associated with human disease in Europe in case reports [5]. Although the number of case reports describing European human infections and disease associated with these agents is accumulating, the evidence for causality of infection with these microorganisms and human disease has not been fully and in vitro propagation of B. miyamotoi [39] and the cultivation of N. mikurensis has only been described in the literature recently [40,41]. Although serological or PCR-based methods have been shown to be useful as diagnostic tools for infectious diseases, they provide only limited evidence for (active) infection [42][43][44]. Thirdly, I. ricinus is frequently infected with more than one microorganism, enabling human exposure and possibly infection with multiple microorganisms, referred to as co-infections, after a singular or consecutive tick-bite. This could confuse the identification of the causative agent of the disease. For example, erythema migrans (EM), a typical manifestation of Lyme borreliosis, has been reported in patients in which infection with N. mikurensis or B. miyamotoi was detected by molecular methods [45,46]. This does not mean that these microorganisms are able to cause EM; it is much more likely that the EM is caused by an infection with B. burgdorferi s.l. and that the other microorganisms were present in the blood, either giving rise to other symptoms or indolent. Furthermore, the detection of R. helvetica and R. monacensis DNA and antibodies in patients with or suspected to have Lyme neuroborreliosis is a puzzling phenomenon and it remains to be elucidated whether coinfection with these spotted fever Rickettsiae has any clinical implications at all [24,25]. Fourthly, a final level of complexity is that some of these other I. ricinus-borne microorganisms might be able to cause disease only under specific conditions, i.e., when a patient is immunocompromised or has a co-infection.
Therefore, the aim of this literature study was to evaluate the published European case reports on tick-borne diseases-other than Lyme Borreliosis or TBE-in order to identify which clinical symptoms are associated with probable infections. This information, as well as data from epidemiological and experimental studies, was used to assess whether there is evidence for a causative relation between infection with these tick-borne microorganisms and human disease in Europe. The literature search was limited to the decade between 01-01-2008 and 01-03-2018, in order to assess the current situation. Our study focused only on microorganisms found in the European I. ricinus ticks and molecularly proven to be transmitted to their human host after a tick-bite, and not on microorganisms found in I. scapularis or I. persulcatus [6].

Literature Search
Our literature search resulted in 3366 unique titles, out of which 3303 did not meet our search criteria based on the abstracts (Table S1), thus yielding 63 papers. In addition to the literature search, we manually included four additional case studies as part of a structural search. These, in total, 67 remaining papers were screened by reading the full text and 60 published case studies were found relevant for this study ( Figure 1). These 60 European case studies, from between January 2008 and March 2018, described 77 individual cases. A. phagocytophilum case reports were most common, accounting for 23 of the papers and 32 cases. This was followed by 20 Babesia case reports describing 21 cases, of which 11 were associated with B. divergens, while seven and three were suspected of B. microti or B. venatorum infection, respectively. A total of 8 publications described 14 N. mikurensis cases, while 6 publications described 3 and 4 cases suspected of R. helvetica or R. monacensis infection, respectively. Finally, three B. miyamotoi cases were described in three publications. Infection with the various I. ricinus borne microorganisms was confirmed by molecular biology methods such as PCR, serology, and microscopy (Table 1).

Clinical Presentation
The most common symptoms reported in the 32 European cases of anaplasmosis analyzed  were fever, headache, malaise, and myalgia (41%-91%). Arthralgia and erythematous skin manifestations such as a maculopapular rash were less common (25% and 22%, respectively), and fewer cases presented with lymphadenopathy, chills, rigors, abdominal complaints, anemia, splenomegaly, and darkened urine (3%-19%) ( Table 2). General laboratory findings included elevated C-reactive protein (CRP) values, leukocytopenia, thrombocytopenia, elevated liver enzymes and lactate dehydrogenase (LDH) values (Suppl. data). One patient was immunocompromised, and hospitalization was required in 88% of cases. Most patients (94%) received antibiotic treatment mainly in the form of doxycycline and subsequently recovered. In two cases, the outcome was fatal.

Exposure to Ticks
Eleven (34%) patients recalled a tick-bite, and 12 (38%) of the cases reported exposure to ticks.

Culture from Ticks or Patient Materials
Although A. phagocytophilum was not cultured in any of the case reports analyzed in this study, culture in a HL-60 promyelocytic cell line from a European patient has been successfully performed in the past in the Czech Republic [70].

Pathology
Both the European and American variants of A. phagocytophilum have been shown to infect human and animal host reservoir neutrophils, hence the name Human Granulocytic Anaplasmosis (HGA) [42,71,72]. The disruptive effect on infected neutrophils may lead to detrimental effects on the host's immune response and poor regulation of inflammatory processes. This is believed to give rise to secondary pathological findings such as tissue damage in the spleen, liver, and kidneys [71]. In farm animals, such as sheep and cattle, the severity of the symptoms was affected by factors such as the host immune status or pre-existing infections [73]. In the cases evaluated in this study, the primary damage to neutrophils and secondary organ damage can be deducted from the reported laboratory test results such as leukocytopenia and elevated liver enzymes, respectively (Table 2). Moreover, bacteria were visualized in 25% of cases in Giemsa stained blood or bone marrow smears.

Transmission Experiments
Transmission of a Dutch strain of A. phagocytophilum from infected I. ricinus to naïve dogs has been shown, as well as the acquisition of the bacteria by I. ricinus in vitro [74].Moreover, a longitudinal study on sheep-to-tick transmission efficiency under natural conditions found molecular evidence of I. ricinus infected with A. phagocytophilum after feeding on their sheep host [75]. Moreover, the bacterium has been detected in I. ricinus ticks as well as animal reservoirs in multiple European countries [76][77][78][79]. Moreover, human tick-bite studies have shown an association between a tick-bite by I. ricinus ticks and the presence of A. phagocytophilum in patient blood. For example, a study in which 626 blood samples of Dutch patients who reported a tick-bite, were analyzed for the presence of tick-borne microorganisms found five samples positive for A. phagocytophilum by molecular screening [6]. Another study in Poland, in which whole blood, serum, and cerebrospinal fluid (CSF) samples from 118 symptomatic patients who reported a tick-bite were screened for tick-borne microorganisms, reported molecular detection of A. phagocytophilum in 14 of the samples [80]. These data support the assumption that this microorganism can be transmitted from I. ricinus to both animals and humans.

Clinical Presentation
In the seven cases where B. microti was reported as the probable cause of disease [81][82][83][84][85][86][87], fever was reported for all patients, anemia in all except one, and also malaise, chills, headache, and abdominal complaints were relatively common (ranging from 57% to 43%). Skin manifestations were reported only in one case ( Table 2). General laboratory findings included elevated CRP, thrombocytopenia, elevated liver enzymes, bilirubin, and LDH (Suppl. Data). Hospitalization was required in 86% of the cases. No fatal cases were described between 2008 and 2018. All patients received treatment with a combination of antibiotics and antiparasitic medication such as azithromycin-atovaquone or proguanil and quinine. It is important to note that infection with B. microti was acquired outside of Europe, namely North America and Uruguay, and solely diagnosed and/or treated in Europe.
In ten additional publications [88][89][90][91][92][93][94][95][96][97], 11 babesiosis cases were reported where infection with B. divergens was the probable cause of disease, and fever was the most common symptom (82%). Anemia, malaise, jaundice, abdominal complaints and darkened urine were all relatively common (ranging from 45% to 27%), while headache, myalgia and arthralgia were reported for only two patients ( Table 2). General laboratory findings included elevated CRP values, leukocytopenia, thrombocytopenia, and elevated liver enzymes, bilirubin, LDH and kidney function (Table 1 and Suppl. data). Hospitalization was required in 91% of the 11 cases, 64% were immunocompromised, out of which 54% were splenectomized. Treatment was administered to 91% of patients. In one case, doxycycline was provided while most followed the guideline advised dual therapy with an antibiotic and antiparasitic medication. Of the nine cases that received combination-therapy, three were fatal, two of which were immunocompromised. Additionally, an immunocompromised patient without treatment died.
Finally, three cases were associated with B. venatorum infection [98][99][100]. Symptoms included anemia (100%), fever, darkened urine (67%), rigors and myalgia (33%). General laboratory findings included elevated CRP values, to a lesser extent thrombocytopenia, and elevated bilirubin, LDH, and kidney function. (Suppl. data). One case required hospitalization, and in all cases, treatment with quinine and clindamycin or azithromycin and atovaquone was followed by recovery. All three patients were immunocompromised and had undergone splenectomy.

Exposure to Ticks
In B. microti suspected cases, patients recalled a tick-bite in 14% of the cases, and exposure to ticks was reported in the remaining 86% of the cases. For cases where B. divergens infection was detected, patients recalled a tick-bite in 45% of cases and exposure to ticks was reported in the other 55% of cases. Exposure to ticks was reported in 33% of the B. venatorum suspected cases.

Culture from Ticks or Patient Materials
None of the Babesia species were cultured in the case reports analyzed. However, B. divergens has been successfully cultured, propagated, and sequenced from samples deriving from a French patient and it has also been cultivated from European cattle [101][102][103]. B. microti has been isolated from field voles in Germany, and B. venatorum has been cultured from roe deer erythrocytes in France [104,105].

Pathology
Babesia spp. are intraerythrocytic parasites, and symptoms such as anemia, jaundice, and hemoglobinuria (presenting as darkened urine) are a result of hemolysis [106].

Transmission Experiments
A European strain of B. microti was transmitted in vivo from I. ricinus to gerbils and vice versa [107]. B. divergens was transmitted to I. ricinus ticks through feeding on inoculated gerbils and cattle [108]. Moreover, B. venatorum was transmitted to I. ricinus ticks fed on infected sheep blood and vice versa [109]. These findings demonstrate that I. ricinus is a competent vector for all three Babesia species.

Borrelia miyamotoi Clinical Presentation
Three European cases with suspected B. miyamotoi disease (BMD) were included in this study [110][111][112], of which two patients were immunocompromised. Symptoms reported included neurological complaints in the two immunocompromised cases due to meningoencephalitis and headache (67%). Fever, abdominal complaints, myalgia, arthralgia, weight loss, and lymphadenopathy were observed in one case (33%). The two immunocompromised patients received antibiotic treatment (ceftriaxone) and recovered. The remaining patient recovered without treatment and showed elevated CRP values, leukocytopenia, and thrombocytopenia.

Exposure to Ticks
Patients recalled tick-bites in all three cases.

Culture from Ticks or Patient Materials
B. miyamotoi was isolated and cultivated in modified Kelly-Pettenkofer medium supplemented with 10% fetal bovine serum from I. ricinus egg masses resulting in two Dutch isolates, of which one was propagated and both were sequenced [113].

Pathology
As opposed to the hematogenous disease commonly seen in immunocompetent cases, in immunocompromised patients, BMD has been reported to manifest neurologically (e.g., meningoencephalitis) [114]. However, recently (after the study period of this review) two new neurological BMD cases have been diagnosed in Sweden, of which one case was immunocompetent [115]. Neurological involvement is demonstrated in the CSF by pleocytosis, visible spirochetes by dark-field microscopy and by molecular analysis specific for relapsing fever Borrelia and Borrelia miyamotoi [115]. Pleocytosis and visible spirochetes in the CSF by dark-field microscopy were also reported in the Dutch immunodeficient case included in this study [110,114].

Transmission Experiments
A Dutch tick isolate of B. miyamotoi has been shown to be infective to I. ricinus fed on infected CD1 mice and vice versa (unpublished) [116]. Moreover, field-collected B. miyamotoi infected I. ricinus larvae have been shown to transmit the spirochete to naive rodents [117]. This suggests I. ricinus is a competent vector for B. miyamotoi transmission.

Exposure to Ticks
Five (36%) patients recalled a tick-bite and two (14%) of the cases reported exposure to ticks.

Culture from Ticks or Patient Materials
Only recently, N. mikurensis was reported to be cultivable in human primary endothelial cells derived from skin microvasculature as well as pulmonary arteries and in the I. ricinus IRE/CTVM20 cell line. Cultivation was performed using PCR-positive blood from Swedish patients [41].

Pathology
The pathology of neoehrlichiosis is not yet fully understood. As N. mikurensis was isolated and propagated in human primary endothelial cells and was visualized in the cytoplasm of circulating endothelial cells of infected patients, it has been suggested that endothelial cells are the infectious target for the bacteria [41]. There is evidence in reported cases for vascular events, such as deep vein thrombosis, thromboembolic events, and transitory ischemic accidents [124]. Moreover, two of the cases in this study presented with arterial aneurysms [119,122].

Transmission Experiments
N. mikurensis has been widely molecularly detected in I. ricinus in multiple European countries, such as Belgium, France, Germany, The Czech Republic, Portugal, Poland, The Netherlands, and Sweden [125][126][127][128]. Moreover, the bacterium has been isolated from xenodiagnostic I. ricinus larvae fed on infected rodents, further implicating this tick species serves as a vector for the bacteria [129]. As far as we are aware, no studies have been published on tick transmission in animal models.

Spotted Fever Rickettsiae Clinical Presentation
Three R. helvetica case reports were included [22,130,131]. All these cases developed fever and headache. Accompanying photophobia was common (67%), while skin manifestations (such as a macular rash), myalgia and arthralgia, were presented in only one case. Laboratory findings included elevated CRP values, elevated erythrocyte sedimentation rate (ESR), leukocytopenia, thrombocytopenia, and elevated liver enzymes and kidney function (Suppl. data). The three cases required hospitalization and recovered following antibiotic treatment with doxycycline.
In the four cases where infection with R. monacensis was reported as the probable cause of disease [132][133][134], skin manifestations (such as erythematous rash and EM) were reported. One of the cases, in which the EM was reported, was found to be co-infected with B. afzelii. Fever and headaches were common symptoms (75%) and anemia was reported in one patient. Laboratory findings included leukocytopenia, as well as elevated ESR and bilirubin (Suppl. data). One patient required hospitalization, but all patients recovered following antibiotic treatment with doxycycline and in one case with azithromycin.

Exposure to Ticks
No tick-bites were reported in the R. helvetica cases, however, all patients had been exposed to ticks. Of the R. monacensis cases 50% reported a tick-bite and 25% were exposed to ticks.

Culture from Ticks or Patient Materials
R. helvetica has been cultivated in African green monkey kidney fibroblast cells (VERO) from CSF of two Swedish cases included in this study [22,131], of which one was also infected by Herpes simplex virus. The bacterium has also been successfully cultured and sequenced from questing I. ricinus ticks in VERO cells [135,136]. R. monacensis was isolated from a patient's blood in Spain and cultivated in VERO cells [132]. Moreover, isolation of R. monacensis from I. ricinus ticks in VERO, mouse fibroblast (L-929), and tick (IRE11, DAE100, ISE6) cell lines have been reported [137].
Pathology R. helvetica DNA has been detected by using a semi-nested PCR of the 16S rRNA and 17-kDa outer-membrane protein genes, in the pericardium and in a lymph node from the pulmonary hilum in one patient and in a coronary artery and the heart muscle of another, in two Swedish cases of sudden cardiac death postulated to be due to a R. helvetica infection. The bacteria were also visualized in macrophages and endothelial cells using Gimenez and May-Grünwald-Giemsa (MGG) staining, wherein their structure was confirmed by transmission electron microscopy [138]. R. helvetica has also been visualized in granulomatous endothelial tissue and macrophages from Swedish patients with sarcoidosis, even though an association with the disease was not established [139]. Additionally, R. helvetica DNA has been detected in CSF of patients in The Netherlands and Denmark [24,140]. In two Danish cases, R. helvetica was cultured from CSF, leukocytopenia was observed, and a macular rash was reported for one of the patients, supporting the findings above. The pathology of R. monacensis has not been studied, but the rashes described in the case reports analyzed, suggest endothelial tissue tropism.  Both R. helvetica and R. monacensis have been repeatedly detected by molecular means both from I. ricinus ticks and, to a lesser extent, from wildlife in various European countries [137,[141][142][143][144][145][146][147][148][149]. As far as we are aware, there are no published transmission experiments in laboratory animals for either species.

Discussion, Conclusions and Knowledge Gaps
Since Koch's postulates are not applicable to all potential disease agents, alternatively, the sum of various alternative approaches could provide sufficient evidence to support causality between a microorganism and human disease, in this case, a tick-borne disease. Such has been the succession of events for several established tick-borne pathogens, for example, for B. burgdorferi s.l. and TBEV. Case reports can provide extensive qualitative, but not quantitative data, thus showing circumstantial, but insufficient scientific evidence for disease causality. The cases presented are usually exceptional ones, such as immunocompromised patients, and because the studies are retrospective in nature, the reports suffer from reporter bias. As part of the current study, by supplementing the data from case reports on A. phagocytophilum, B. venatorum, B. divergens, B. microti, B. miyamotoi, N. mikurensis, R. helvetica, and R. monacensis with epidemiological and experimental studies, we have assessed their causal relationship to human disease. Thus, we found that to varying degrees, the existing evidence is incomplete for all the microorganisms mentioned above, but seemed strongest for A. phagocytophilum and B. divergens. This approach also provided a comprehensive outlook on the current knowledge gaps that need to be filled (Table 3).  A. phagocytophilum causes illness in immunocompetent patients in Europe, and the case reports amounted to half of all papers included in this review. The clinical picture, laboratory test results, and low fatality rate were reminiscent of what has been described for HGA in the USA [26,43]. A rash was reported in a minority of patients, which is not considered to be a specific sign of HGA and may point to co-infections with other tick-borne bacteria, such as Rickettsia or Borrelia spp. [134,[153][154][155], as was confirmed for one of these cases [56]. In Europe A. phagocytophilum has been cultivated from a patient's blood and its tissue tropism, namely neutrophils, has been demonstrated [42,70]. A. phagocytophilum has not been isolated from I. ricinus. However, there is ample evidence of its association with I. ricinus ticks [76,77] and evidence that this tick species can be infected by natural reservoirs [75].
In accordance with published data [156,157], most cases reporting Babesia infection in Europe were attributed to B. divergens, and, while disease has been associated with immunocompromised and splenectomized patients [157], almost half of the cases were immunocompetent. Four cases were fatal, and three of these were immunocompromised and one was immunocompetent and had a co-existing Lyme borreliosis infection [90]. Co-infections with Babesia are a known possibility in Lyme borreliosis patients and should be considered in cases from endemic regions presenting with high fever, anemia, thrombocytopenia, leukocytopenia, or other hemolysis signs, or in cases who do not respond to treatment [158]. Co-infection of B. microti and B. burgdorferi s.l. has been found to increase both the severity and duration of illness in North America [153,159,160], such correlations have not been shown for co-infection with B. divergens and B. burgdorferi s.l. Our analyses also suggested that B. microti is able to cause disease in Europe, but mainly as an imported agent presenting as a non-fatal illness in immunocompetent patients. Although it is present in I. ricinus and European wildlife and has been detected in human samples from Germany, Poland, and Switzerland [157], the number and severity of B. microti cases, be it from lack of awareness or genetic difference, is low when compared to North America [157]. In the USA, 2358 cases were reported in 2017 and fatality rates of up to 6%-9% have been reported in immunocompetent patients [156,161]. The first European originated case, probably acquired by a blood transfusion, was published in 2007 [162], however, the protozoa have not been cultivated from European patients or I. ricinus. Cultivation of European strains would facilitate genomic analyses in the search for differences in pathogenicity when compared to North American strains. The few B. venatorum cases reported were relatively mild and affected immunocompromised patients. Babesia venatorum can be transmitted by I. ricinus in laboratory settings [109]. However, cultivation from ticks and patients remains to be performed and would also assist the development of specific serological tests for diagnostic purposes and future population studies.
Borrelia miyamotoi is an emerging tick-borne microorganism whose potential to cause disease has only recently been disclosed [114]. Since the first case reports in 2011 in Russia, over 450 cases have been published, mainly from Asia and North America, with a total of 5 cases from Europe. B. miyamotoi is able to cause an acute febrile disease with flu-like symptoms. Skin lesions, such as EM, have been reported in B. miyamotoi suspected cases, but are presumed to be related to a co-infection with the etiologic agent of Lyme disease, B. burgdorferi s.l. In immunocompromised cases, of which most originate from Europe, the disease seems to develop neurologically, however, a meningoencephalitis in an immunocompetent case has been reported. Nevertheless, more European cases need to be evaluated to assess the full extent of the clinical symptoms and effectiveness of the available diagnostic modalities. B. miyamotoi has not been cultivated from an individual infected by an I. ricinus tick-bite. However, it has been cultivated from 12 Russian patients, some from European Russia, of which 6 were sequenced and published [163], with I. persulcatus being the most likely vector. Cultivation of more B. miyamotoi isolates from I. ricinus and European human cases would enable comparative genomic analyses between isolates from Europe, North America, and Asia [164]. This could be an enormous impetus for basic, molecular, and epidemiological research and could lead to the development of specific diagnostic tools for this emerging potential TBP.
Another emerging potential TBP is N. mikurensis [124]. The findings of this study support the growing evidence of N. mikurensis as a disease agent affecting the immunocompromised population, and show it is also found in immunocompetent patients. EM was reported in two cases where co-infection with A. phagocytophilum in one and B. burgdorferi s.l. in the other was present [46]. A Norwegian study, in which EM patients were screened by PCR for the presence of N. mikurensis, reported seven positive samples, of which only three were positive for B. burgdorferi s.l. by serology [165]. However, this could be explained by the low diagnostic accuracy of serology in early Lyme borreliosis [166]. Three other cases analyzed in this study, of immunocompromised patients presenting with a rash, were diagnosed by a positive N. mikurensis PCR, and in two cases by transmission electron micrographs. However, no serology was performed in order to rule out a co-infection that could have caused the rash [120,121]. Although Ehrlichia species are known to infect granulocytes or mononuclear phagocytes [167], the bacterium has been cultivated from patient's blood in a tick cell line and in human primary endothelial cells [41]. Routine cultivation, as well as cultivation from ticks would enable development of the serological assays, which are currently lacking from the routine practice.
Consistent with it being a spotted fever group Rickettsia, three R. monacensis cases presented with a rash, and in one case in which an EM was present, a co-infection with B. afzelii was detected [134]. However, no studies have been performed to elucidate the specific pathology of this microorganism. The clinical picture reported for R. helvetica in this study included headaches and photophobia, which is suggestive of neurological involvement. One of the cases presented with subacute meningitis [22] and although the diagnosis of neuroborreliosis was rejected based on serology, the test was performed in an early disease stage when test sensitivity infamously falls short. Another case presented with meningoencephalitis [131] and was positive for Herpes Simplex Virus 2, which could account for the clinical picture. In the three case reports included in the study R. helvetica was either isolated and cultivated or isolated, accompanied by seroconversion indicating that this microorganism can cause infection in humans. However, proof found of a Rickettsia infection does not imply or translate to symptomatic disease. Therefore the evidence should not be mistaken for disease causation, which has not been demonstrated [22,131]. Interestingly, R. helvetica has been detected as a co-infection with Lyme neuroborreliosis both in patient blood and CSF in a Dutch study [24]. R. helvetica has also been found as a co-infection in Danish Lyme borreliosis and neuroborreliosis patients [140], however the involvement of this bacterium with neurological symptoms has not been studied. Although both R. helvetica and R. monacensis have been isolated from patients and I. ricinus ticks, only few case reports have been published on cases caused by these species and no transmission experiments have been performed to date, in contrast to, for example, R. conorii, an established human pathogen in Europe.
In all case reports analyzed in this study, diagnostic tools such as microscopy, serology, and molecular analysis were required in order to reach a diagnosis. In some cases, these tools were employed retrospectively, as they were not accessible in routine practice. Moreover, the cultivation of microorganisms such as A. phagocytophilum, N. mikurensis, and Babesia spp. was not attempted as their growth requires dedicated settings, is technically demanding, and time-consuming. Even in experimental settings, cultivation of these microorganisms can be arduous. To date, cultivation from I. ricinus has been reported only for B. miyamotoi, R. helvetica, and R. monacensis [113,135,137] and cultivation from I. ricinus acquired cases has not been performed for B. venatorum, B. microti, and B. miyamotoi.
The additional evidence needed in order to conclusively appraise disease causality and elucidate the impact of I. ricinus transmitted infections in Europe for these tick-borne microorganisms can be achieved by the fulfillment of several steps. Firstly, to enhance our understanding of the tissue tropism and specific pathology of microorganisms, such as R. helvetica, R. monacensis, and N. mikurensis, experimental transmission experiments using laboratory animals should be performed. Secondly, new and specific serological tools should be developed for the diagnosis of I. ricinus-borne microorganisms. This could be facilitated by cultivation of the tick-borne microorganisms from patients or ticks, followed by next-generation sequencing and bioinformatic analyses, for the discovery of new potential antigens. Thirdly, prospective studies need to be performed including patients with febrile illness after a tick-bite, the common denominator in the clinical manifestation of the different tick-borne microorganisms.

Literature Search
The literature search was performed using the Pubmed and Embase databases (Table S1). In addition, a structural search was performed in order to find additional cases and information.

Selection Criteria
The search focused on suspected disease cases with Anaplasma phagocytophilum, Babesia divergens, Babesia microti, Babesia venatorum, Borrelia miyamotoi, Neoehrlichia mikurensis, Rickettsia helvetica, and Rickettsia monacensis. Case reports and series of patients diagnosed in Europe, published in the period between 01-01-2008 and 01-03-2018, were included. The process was performed independently by TA and DH, and when disagreements occurred, the study was re-evaluated by both TA and DH under the supervision of HS, KK, and JH. Publications written in English, Dutch, and Spanish were included.

Data Extraction
Parameters extracted for all case reports/series were: Sex, age, immune status, underlying health conditions, pre-existing treatments, history of a tick-bite or exposure to ticks, clinical symptoms, general laboratory findings, hospitalization, treatment, disease outcome, diagnostic evaluation, excluded causes of disease and co-infections (Table S2-S9). Within clinical symptoms, "fever" was attributed to a case whenever the measured temperature exceeded ≥ 37.5°C (99,5 o F) [168] or when the authors reported fever as a symptom. History of a tick-bite was recorded whenever a patient recalled a tick bite on presentation. Exposure to ticks was recorded whenever a patient worked in a high-risk occupation, e.g., forest ranger, had engaged in activities considered to be high risk for a tick bite such as hiking, or had been exposed to animals.

Data Analysis
For each tick-borne microorganism, cases were evaluated for evidence of a causal relationship between the microorganism and the disease described by establishing the nature and frequency of five criteria: The clinical presentation, association to tick-bite(s), culture from ticks or patient materials, transmission experiments, and the ability to induce specific pathology. In addition, for the analyses on cultivation from ticks or patients, transmission experiments and pathology, information was added by selectively searching the literature.

Conflicts of Interest:
The authors declare no conflict of interest.