**Preface to "***Babesia* **and Human Babesiosis"**

*Babesia* is a genus of intraerythrocytic protozoan parasites belonging to the exclusively parasitic phylum Apicomplexa. There are more than a hundred known species of this genus, occurring mainly in mammals, but also in birds, and all transmitted by ticks, which are blood-sucking arthropods related to spiders. Ixodid (hard-bodied) ticks are vectors of the vast majority of *Babesia* spp., but a small number of babesias are transmitted by argasid (soft-bodied) ticks. For many years, *Babesia* spp. were only known as important parasites of domestic animals and were the first pathogens shown to be transmitted by an arthropod vector when, in 1893, Smith and Kilborne reported the vector role of cattle ticks in redwater fever (babesiosis) in the USA [1]. Human babesiosis was first described in 1957 when it occurred as a fulminant and ultimately fatal infection in a Croatian farmer [2]. More human cases followed over the next 50 years, and at least four taxonomically classified *Babesia* species (*B. divergens*, *B. duncani*, *B. microti*, and *B. venatorum*) have now been confirmed as zoonotic pathogens, with some others that have not yet been identified to species.

The main pathological event of infection with these parasites is the destruction of erythrocytes, resulting in haemolytic anaemia with added complications due to the release of toxins and waste products into the bloodstream. Further damage to the host can be caused by cytokine storms as the host's immune system responds to infection. In many respects, the pathology of babesiosis is similar to that of the much better-known disease, malaria, caused by *Plasmodium* spp.

This Special Issue consists of 11 reviews that between them address the global babesiosis situation, the disease in Europe, the history and current status of *B. microti* in the USA, babesiosis in relation to sickle cell anaemia, experimental infections of ticks, transfusion transmission, the significance of major surface antigens, advances in the diagnosis of babesiosis, historical and current approaches to treatment and management, and babesiosis in relation to climate change. Additionally, six research articles are presented addressing the discovery of a new zoonotic genotype of *B. divergens*, the characterisation and function of certain proteins involved in parasite–erythrocyte interaction, the identification of proteases as possible drug targets, the identity of piroplasms in ticks removed from deer in Portugal, and the identification of an alternate growth medium that can support the *in vitro* growth of *B. duncani* in human erythrocytes.

In their review on the worldwide occurrence of human babesiosis, Kumar et al. [Ch. 1] draw attention to the fact that this is an emerging zoonosis, with increasing reports of infections caused by the known zoonotic species in new areas, for example, in China, in addition to cases involving *Babesia* parasites of undetermined species. They conclude that the true number of affected patients is considerably underestimated, particularly in regions where clinical and diagnostic overlap with malaria occurs, and call for improved surveillance and continued research on treatment and prevention. The authors mention climate change as a possible factor in the gradual spread of *B. microti* in the USA, and the role of climate in the epidemiology of zoonotic babesiosis in general is discussed more fully by Gray and Ogden [Ch. 2], with particular reference to climate effects on the vector ticks. While extensive data suggest that global warming is affecting the distribution of the *Ixodes* vectors, no changes in the current occurrence of zoonotic babesiosis can, as yet, be convincingly attributed directly to climate change, though models suggest that this is only a matter of time.

Hildebrandt et al. [Ch. 3] discuss European babesiosis in more detail. Compared with the USA, the disease is relatively rare in Europe, but the authors point out that most cases present as medical emergencies, mainly in immunocompromised patients, and particularly in those that are asplenic. Unusually, an attempt has been made to present data on every recorded case that has occurred in the last two decades, with the hope of shedding new light on both the epidemiology of the disease as well as on diagnosis and management. Most human babesiosis cases in Europe are due to infection with *B. divergens* and *B. microti*, although the true prevalence of the latter is unknown because of the apparent low pathogenicity of European strains of this parasite. There is also uncertainty about the epidemiology of the genuinely pathogenic *B. divergens*, particularly the possible role of red deer as reservoir hosts. This topic is again addressed in a research paper by Fernandez et al. [Ch. 4], who describe a study in which ixodid ticks removed from deer in a Portuguese nature reserve were analysed for piroplasm infections. *B. divergens* sequences were detected that were apparently identical to those associated with human and bovine babesiosis, and it is concluded that the most likely source of these parasites was the deer. Other interesting findings include the association of *Theileria* spp. with *Ixodes ricinus* and the abundant occurrence of an exophilic form of the brown dog tick, *Rhipicephalus sanguineus*.

*Babesia microti*, the causal agent in the vast majority of cases, particularly in the USA, is the subject of two reviews. Telford et al. [Ch. 5] describe in detail the emergence of this pathogen 50 years ago. This is probably the first time that all the salient facts behind the appearance of this pathogen and the subsequent spread of *B. microti* babesiosis in the USA are presented in detail, which will make interesting and enlightening reading for all babesiologists. A range of interventions are described, and although the extent of their implementation has proved disappointing, the authors remain optimistic that by the centennial of the discovery of "Nantucket fever", technological advances will have resolved many of the control and prevention problems. In the second review on *B. microti*, Goethert [Ch. 6] describes its worldwide diversity, knowledge of which has evidently increased markedly over the decades since the parasite's emergence as a human pathogen. The author argues that because many of the studies on *B. microti* were conducted before the availability of molecular analysis, an understanding of its ecology has been hampered by confusion about parasite identity. *B. microti* has now been taxonomically allocated to five distinct clades within the species complex, but problems with identity evidently persist in some recent studies.

Parasite diversity has also drawn the attention of researchers in the study of *B. divergens*-like pathogens since the occurrence of four human cases in the USA [3, 4, 5, 6] and two in Europe [7, 8]. In some of these reports, the infectious agent was initially identified as *B. divergens*, but subsequent analysis has established that they are all clearly distinct from this species and are currently described as *B. divergens*-like or have been given an abbreviation to indicate the location of the case. Thus, the causal agent of the first of these [3] occurred in Missouri and is described as *Babesia* sp. MO1. In this Special Issue, Bonsergent et al. [Ch. 7] describe an isolate obtained from a case in France, which caused a relatively mild infection, compared with classic *B. divergens* babesiosis. The subsequent molecular analysis determined that the parasite involved, which they name *Babesia* sp. FR1, belongs to the MO1 clade. Their study demonstrates that variations in the severity of suspected *B. divergens* babesiosis [9] may be due to infections with *B. divergens*–like parasites rather than with the classic *B. divergens* of cattle. The reservoir host of *Babesia* sp. MO1 is believed to be the cotton-tail rabbit (S*ylvilagus floridanus*), and while the reservoir host of *Babesia* sp. FR1 is unknown, the European rabbit (*Oryctolagus cuniculus*) is implicated by its high abundance in the habitat where the infection is thought to have been contracted.

The list of zoonotic *Babesia* spp. is gradually lengthening, but it is difficult to determine the tick vector involved in the transmission of parasites known only as isolates from patients. The detection of parasite DNA in ticks is only indicative of vector status and absolute proof requires experimental demonstration of transmission under controlled conditions [10]. A review of the approaches and technologies to achieve such proof is presented by Bonnet and Nadal [Ch. 8], who discuss the application of ticks to both naturally infected and experimental animals, and also the increasing use of artificial tick-feeding systems. They conclude that systems for the experimental infection of ticks are vital tools for the determination of vector competence, enhancing our knowledge of pathogen ecology and of *Babesia* spp. life cycles, and that consideration should be given to the standardisation of artificial-feeding protocols.

Although tick transmission is the primary means by which *Babesia* spp. infect humans, blood transfusions are an increasingly important source of infection, particularly of *B. microti* in the USA. Bloch et al. [Ch. 9] review the history of transfusion-transmitted babesiosis, mainly in the USA, evaluate the evolution of surveillance, assay development, and screening policy in the USA, and suggest that the current American model for the prevention of transfusion babesiosis could form the basis for similar measures in other countries where the perception of transfusion transmission risk is currently low. One of the groups of patients that is particularly prone to haemolysis and requires frequent blood transfusions are those suffering from haemoglobinopathies such as sickle cell anaemia and thalassaemia. Little is known about the course of babesiosis in such patients, but it has been accepted for many years that haemoglobinopathies afford some protection against malaria, and studies on *Babesia* spp. in this context, reviewed here by Beri et al. [Ch. 10], suggest that such conditions also hinder intraerythrocytic growth of parasites. Possible mechanisms for the resistance of sickle cells to *Babesia* spp. are explored and suggestions are made for further studies to identify the possible "Achilles heel" of both *Babesia* and *Plasmodium* spp. that could result in effective interventions.

The detection of *Babesia* parasites in stored blood by molecular methods is an essential component of screening procedures for blood transfusion and is also the most reliable approach for detection of parasites in clinical cases when parasitaemias are low, whereas microscopy in the hands of experienced laboratory staff is useful at higher parasitaemias. Meredith et al. [Ch. 11] address the history, current status, and future prospects for laboratory diagnosis of *B. microti*, with particular emphasis on the application of modern technologies such as exploitation of the CRISPR–Cas system, which markedly increases the sensitivity of nucleic acid test systems. Serological testing for babesiosis has mainly relied on immunofluorescence techniques to detect surface antigens, and increased knowledge of the nature of these surface antigens is important. Delbecq [Ch. 12] reviews the major surface antigens of *B. microti* and *B. divergens*, highlighting their role in both erythrocyte invasion and the immune response. He concludes that the increased knowledge of the major antigens will contribute to the development of vaccines, and of more sensitive serological assays and antigen capture assays that could be used to identify biomarkers for exposure, active infection, and protection. Other antigens, members of the rhoptry-associated protein-1 (pRAP-1) family, are the subject of a work by Bastos et al. [Ch. 13]. These proteins are secretory products of the apical complex in piroplasms, which plays an essential role in cell invasion by the parasite. Rhoptry proteins have not received the attention they should and the study described here suggests the involvement of pRAP-1 in parasite adhesion, attachment, and possibly evasion of the immune response. Antibodies in *B. microti-*infected humans recognise recombinant forms of the two proteins studied, suggesting that they could be candidates for both diagnostic assays and vaccines.

Efficacious drug treatment of patients is central to the management of babesiosis and a review of antimicrobial use in the past and present by Renard and Ben Mamoun [Ch. 14] draws attention to the fact that the currently available drugs are limited and have been repurposed rather than developed specifically as antibabesials. Since they are associated with either significant side effects or the rapid emergence of drug resistance, it is clear that new therapeutic strategies are required. *In vivo* models for antibabesial evaluation using mice, hamsters, and gerbils have been available for some years but continuous culture *in vitro* has been restricted to *B. divergens* up to the present. However, Singh et at., have demonstrated that the DMEM-F12 medium supports the continuous *in vitro* culture of *B. duncani* in human erythrocytes [Ch. 15]. This finding in combination with the development of the 'in culture-in mouse' (ICIM) model of *B. duncani* infection, also conducted by Ben Mamoun's laboratory [11], are major advances and are likely to result in *B. duncani* becoming the species of choice for the discovery of antimicrobials against all the zoonotic *Babesia* spp.

*Babesia microti* is the predominant zoonotic species and is also arguably the least susceptible to existing antimicrobials [12]. The identification of chemotherapeutic targets in these parasites thus becomes an important research priority. Florin-Christensen et al. [Ch. 16] focus on species-specific proteases and have used bioinformatics to identify genes in the *B. microti* genome that code for these enzymes. They classify 89 proteases into five groups and report that comparisons between *B. microti* and *B. bovis* reveal differences between sensu lato and sensu stricto parasites, reflecting their distinct evolutionary histories, which is probably relevant to their susceptibilities to antibabesials [12]. In another work on proteases [Ch. 17] Snebergerov ˇ a et al. investigate aspartyl proteases in ´ *B. microti*, particularly in relation to homologues of known function in other parasites, such as plasmepsins in *Plasmodium* spp. They suggest that analogies with plasmodial plasmepsins indicate piroplasmid aspartyl proteases as potentially important therapeutic targets.

We hope this Special Issue will motivate research scientists to further develop strategies for the prevention and control of babesiosis in the future. Improvements are required in diagnosis, the rigorous typing and identification of *Babesia* parasites, prevention of transfusion transmission, and the discovery of novel antibabesial drugs. The development of safe and effective vaccines for use in humans remains an unrealised goal and is an important research priority.

The researchers who have participated in this Special Issue remind us that zoonotic babesiosis is a complex emerging disease, in which ticks and domestic and wild animals have crucial roles so that environmental factors, particularly in a climate change context, must be taken into account. In the coming years, multidisciplinary collaboration between research groups, the use of digital tools for analysing and sharing essential data about current and new species, the involvement of health authorities in the implementation of surveillance systems, and the development of specific funding strategies for emerging infections such as babesiosis, will be decisive in achieving the necessary goals. Finally, it is imperative to inform and collaborate with veterinary scientists, community health care workers, and the general population in order to determine and reduce the risk of zoonotic babesiosis.

Our sincere thanks to all the authors for their excellent contributions to this book and to the Special Issue assistant Editor, Anne Wang, for her assistance throughout.

#### **References**

1. Smith, T.; Kilborne, F.L. Investigations into the nature, causation and prevention of Southern cattle fever.*In Ninth Annual Report of the Bureau of Animal Industry for the Year 1892*; Government Printing Office: Washington, DC, USA, 1893; pp. 177–304.

2. Skrabalo, Z.; Deanovic, Z. Piroplasmosis in man; report of a case. *Doc. Med. Geogr. Trop*. 1957, 9, 11–16.

3. Herwaldt, B.; Persing, D.H.; Precigout, E.A.; Goff, W.L.; Mathiesen, D.A.; Taylor, P.W.; ´ Eberhard, M.L.; Gorenflot, A.F. A fatal case of babesiosis in Missouri: Identification of another piroplasm that infects humans. *Ann. Intern. Med*. 1996, 124, 643–650.

4. Beattie, J.F.; Michelson, M.L.; Holman, P.J. Acute babesiosis caused by *Babesia divergens* in a resident of Kentucky. *N. Engl. J. Med*. 2002, 347, 697–698.

5. Herwaldt, B.L.; de Bruyn, G.; Pieniazek, N.J.; Homer, M.; Lofy, K.H.; Slemenda, S.B.; Fritsche, T.R.; Persing, D.H.; Limaye, A.P. *Babesia divergens*-like infection, Washington State. *Emerg. Infect. Dis*. 2004, 10, 622–629.

6. Burgess, M.J.; Rosenbaum, E.R.; Pritt, B.S.; Haselow, D.T.; Ferren, K.M.; Alzghoul, B.N.; Rico, J.C.; Sloan, L.M.; Ramanan, P.; Purushothaman, R.; et al. Possible transfusion-transmitted *Babesia divergens*-like/MO-1 infection in an Arkansas patient. *Clin. Infect. Dis*. 2017, 64, 1622–1625.

7. Olmeda, A.S.; Armstrong, P.M.; Rosenthal, B.M.; Valladares, B.; del Castillo, A.; de Armas, F.; Miguelez, M.; Gonzalez, A.; Rodriguez Rodriguez, J.A.; Spielman, A.; et al. A subtropical case of human babesiosis. *Acta Trop*. 1997, 67, 229–234.

8. Centeno-Lima, S.; do Rosario, V.; Parreira, R.; Maia, A.J.; Freudenthal, A.M.; Nijhof, A.M.; ´ Jongejan, F. A fatal case of human babesiosis in Portugal: Molecular and phylogenetic analysis. *Trop. Med. Int. Health TMIH* 2003, 8, 760–764.

9. Martinot, M.; Zadeh, M.M.; Hansmann, Y.; Grawey, I.; Christmann, D.; Aguillon, S.; Jouglin, M.; Chauvin, A.; De Briel, D. Babesiosis in immunocompetent patients, Europe. *Emerg. Infect. Dis.* 2011, 17, 114–116.

10. Gray, J.S.; Estrada-Pena, A.; Zintl, A. Vectors of babesiosis. *Annu. Rev. Entomol*. 2019, 64, 149–165.

11. Pal, A.C.; Renard, I.; Singh, P.; Vydyam, P.; Chiu, J.E.; Pou, S.; Winter, R.W.; Dodean, R.; Frueh, L.; Nilsen, A.C.; et al. *Babesia Duncani* as a model organism to study the development, virulence and drug susceptibility of intraerythrocytic parasites *in vitro* and *in vivo*. J. Infect. Dis. 2022, doi:10.1093/infdis/jiac181.

12. Gray, J.; Zintl, A.; Hildebrandt, A.; Hunfeld, K.P.; Weiss, L. Zoonotic babesiosis: Overview of the disease and novel aspects of pathogen identity. *Ticks Tick-Borne Dis*. 2010, 1, 3–10.

About the cover: This image represents *Babesia divergens* parasites and is based on 3D tomograms obtained by cryo-soft X-ray tomography (cryo-SXT) at the Alba Synchrotron, Barcelona, Spain. Cryo-SXT assays were conducted by Drs. Javier Conesa, Daniel Luque, Javier Chichon, Eva Pereiro, ´ Luis M. Gonzalez and Estrella Montero.

> **Estrella Montero, Jeremy Gray, Cheryl Ann Lobo, and Luis Miguel Gonz´alez** *Editors*

## *Review* **The Global Emergence of Human Babesiosis**

**Abhinav Kumar <sup>1</sup> , Jane O'Bryan 2,3 and Peter J. Krause 1,\***


**Abstract:** Babesiosis is an emerging tick-borne disease caused by intraerythrocytic protozoa that are primarily transmitted by hard-bodied (ixodid) ticks and rarely through blood transfusion, perinatally, and organ transplantation. More than 100 *Babesia* species infect a wide spectrum of wild and domestic animals worldwide and six have been identified as human pathogens. *Babesia microti* is the predominant species that infects humans, is found throughout the world, and causes endemic disease in the United States and China. *Babesia venatorum* and *Babesia crassa*-like agent also cause endemic disease in China. *Babesia divergens* is the predominant species in Europe where fulminant cases have been reported sporadically. The number of *B. microti* infections has been increasing globally in recent decades. In the United States, more than 2000 cases are reported each year, although the actual number is thought to be much higher. In this review of the epidemiology of human babesiosis, we discuss epidemiologic tools used to monitor disease location and frequency; demographics and modes of transmission; the location of human babesiosis; the causative *Babesia* species in the Americas, Europe, Asia, Africa, and Australia; the primary clinical characteristics associated with each of these infections; and the increasing global health burden of this disease.

**Keywords:** babesiosis; *Babesia microti*; epidemiology; immunoepidemiology; case surveillance; babesiosis

#### **1. Introduction**

Human babesiosis is caused by intraerythrocytic protozoal parasites in the phylum Apicomplexa and is transmitted by hard bodied ticks. It is rarely transmitted through red blood cell transfusion, transplacentally from mother to fetus, and through organ transplantation. Babesiosis is an emerging infection with increasing numbers of cases being reported throughout the world (Figure 1) [1–8].

More than 100 species of *Babesia* have been described that infect a wide array of wild and domestic animals [9,10]. Babesiosis is a significant problem for cattle and has had a major economic impact in several cattle producing countries. Six primary species have thus far been confirmed as human pathogens: *Babesia crassa*-like *agent, Babesia divergens, Babesia duncani, Babesia microti, Babesia motasi,* and *Babesia venatorum*. Several other genetically related pathogen substrains have been reported to infect humans, including *Babesia divergens*-like and *Babesia microti*-like pathogens (Table 1).

Human babesiosis is found primarily in the temperate zone. The predominant species is *B. microti*, which is endemic in the northeastern and northern midwestern United States and southwestern China [1,3,4,6]. *B. crassa*-like pathogen and *B. venatorum* are endemic in northeastern China [11,12]. *B. divergens* is found most commonly in Europe [2,5]. Cases of babesiosis have been sporadically reported in Australia [13], Bolivia [14], Brazil [15], Canada [16,17], the Canary Islands [18], Colombia [19], Ecuador [20], Egypt [21], India [22,23], Japan [24], Korea [25,26], Mexico [27], Mongolia [28], Mozambique [8], South Africa [29], Taiwan [30], and Turkey [31] (Table 2).

**Citation:** Kumar, A.; O'Bryan, J.; Krause, P.J. The Global Emergence of Human Babesiosis. *Pathogens* **2021**, *10*, 1447. https://doi.org/10.3390/ pathogens10111447

Academic Editors: Estrella Montero, Jeremy Gray, Cheryl Ann Lobo and Luis Miguel González

Received: 24 September 2021 Accepted: 30 October 2021 Published: 6 November 2021 Corrected: 23 May 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

**Figure 1.** Geographic distribution of major areas of human babesiosis transmission. The map depicts the major areas where human babesiosis has been reported. Additional areas where human babesiosis has been reported but are not shown in the figure are mentioned in the text. Solid colors indicate areas where human babesiosis is endemic. Stippled areas indicate areas where babesiosis is sporadic with ≥10 cases reported. Circles depict areas where 1–10 cases have been reported. Colors distinguish the etiologic agents: *Babesia crassa*-like agent (gray), *Babesia duncani* (orange), *Babesia divergens* (blue), *Babesia microti* (red), *Babesia motasi* (black), and *Babesia venatorum* (green). White circles depict cases caused by *Babesia* spp. that were not characterized. Asymptomatic infections are omitted (adapted from The New England Journal of Medicine, Edouard Vannier, and Peter J. Krause, Human Babesiosis, 2012, 366, 2397. Copyright (2021) Massachusetts Medical Society. Reprinted with permission [1]).



adapted from Puri et al. Frontiers in Microbiology, 2021 [37].

*Babesia* parasites were first described by Victor Babes in Romanian cattle in 1888 [38]. The first human case of babesiosis was described in 1957 by Skrabalo and Deanovic in Yugoslavia and the second in 1968 in California [32,33,39]. The causative *Babesia* species was not determined in either instance. A year later, a third babesiosis patient was reported and the causative species was identified as *B. microti.* The patient was a resident of Nantucket Island in Massachusetts where babesiosis was soon recognized as endemic [40]. Additional cases were reported in the southeastern New England mainland and from there the disease spread eastward, northward, and southward [41–46]. A primary cause of this emergence is thought to be a marked increase in the white-tailed deer population that greatly amplifies the number of vector *Ixodes scapularis* ticks. Other causes include an increase in the human population, home construction in wooded areas, increased recognition of the disease by physicians and the lay public, and improved diagnostic testing [1,39,41]. The emergence of babesiosis has lagged behind that of Lyme disease, even though it is transmitted by

the same tick and is sometimes transmitted simultaneously [45,47]. Babesiosis due to *B. microti* is now endemic from Maryland to Maine and in the northern Midwestern states of Minnesota and Wisconsin. A modest number of cases of *B. duncani* have been reported on the West coast [48]. Babesiosis due to a *Babesia divergens*-like pathogen has been identified in patients in five states: Arkansas, Kentucky, Michigan, Missouri, and Washington [36,49–52].

Babesiosis should be suspected in patients who live in or travel through an endemic area or have received a blood transfusion within the previous six months and present with typical symptoms that include fever, chills, sweats, headache, and fatigue [2,53]. The disease is confirmed by identifying *Babesia*-infected red blood cells on thin blood smear or amplification of *Babesia* DNA using polymerase chain reaction (PCR) [1,2,54–56]. Atovaquone and azithromycin (the drug combination of choice) or clindamycin and quinine treatment are usually very effective, although prolonged illness may occur in immunocompromised hosts with a mortality rate as high as 20% [1,2,11,54,57,58].

In this review we focus on the epidemiology of human babesiosis. We will discuss epidemiologic tools used to monitor disease location and frequency, modes of transmission and demographics, the location of human babesiosis, the causative *Babesia* species in the Americas, Europe, Asia, Africa, and Australia, and the primary clinical characteristics associated with each of these infections.


**Table 2.** World-wide case distribution of human babesiosis \*.

\* The well-established *Babesia* spp. that cause human babesiosis in China, Europe, and the United States are listed. The *Babesia* spp. that have been identified in countries where only a few cases of human babesiosis have been identified in case reports or small case series (<10 cases) are also identified. Some causative agents have not been confirmed in larger case series so are not yet accepted as established causes of human babesiosis. *Babesia* sp. designate where a specific species was not identified.

#### **2. Epidemiologic Tools**

A number of methods are used to determine the frequency, location, and future emergence of infections, as part of local, state, national, and international disease tracking efforts. Case surveillance is of central importance and other methods, including case reports and case series, provide validation of surveillance data.

#### *2.1. Case Surveillance*

Public health officials at the local, state, and national levels collect reports of disease cases from physicians, hospitals, and laboratories. Babesiosis is one of about 120 diseases that are nationally notifiable in the United States and it was so designated in 2011. Case surveillance is of primary importance in helping the United States Centers for Disease Control and Prevention (CDC) determine the location of diseases, the number of cases of diseases at various locations, and the appropriate responses to prevent outbreaks (https: //www.cdc.gov/nndss/about/index.html, accessed on 27 July 2021) [60–62]. Traditionally, case surveillance has been carried out through physician reporting of notifiable diseases. Recent variations on this standard approach include citizen science participation where members of the public collaborate with scientists to collect samples and data [63].

#### *2.2. Case Reports and Case Series*

A case report is a description of a single patient that usually includes symptoms and signs, diagnosis, and treatment. It often describes a new disease but can also describe a novel aspect of a well-known disease. Case reports include descriptions of a previously unreported disease or the presence of an emerging disease in a new location, insights into disease pathogenesis, and generation of new hypotheses or new ideas. Limitations include a lack of generalizability, inability to show cause and effect, potential for overinterpretation of the cause or outcome of disease, and a narrow focus on rare aspects of a disease [64].

A case series involves a report of a group of cases (usually more than three) that can provide information about infection transmission, risk factors for disease, diagnosis, treatment, and outcome of disease. Case series are descriptive in nature rather than hypothesis driven and are prone to selection bias and findings are often not generalizable to other populations. Despite these limitations, the publication of case reports and case series is important to raise awareness of emerging infectious diseases. Indeed, the discovery of the first human case of babesiosis was published as a case report [33]. The first reports of endemic human babesiosis were case series, describing infections due to *B. microti* [65], *B. venatorum* [12], and *B. crassa*-like agent [11].

#### *2.3. Serosurveys*

A serosurvey is a sera screening analysis of a group of people designed to determine the prevalence of infection. Seroprevalence provides a measurement of disease exposure and risk that is based on the antibody response of those tested [60,66–68]. Antibody generally can be detected about two weeks after the onset of infection and may last as little as a year or as long as a lifetime, depending on the infectious pathogen and the immune characteristics of the host. Serosurveys are one of several immunoepidemiologic tools used to improve our understanding of the epidemiology of a disease [69]. They complement case surveillance and have the advantage of capturing both asymptomatic and symptomatic infection [60]. They also inform public health officials of notifiable diseases. Thus, serosurveys are less likely to underestimate the true prevalence of infection than case finding [60,70]. One challenge of serosurveys and case surveillance methods is that antibody assays and case definitions often change over time, altering interpretation of disease trends and incidence of cases [68]. Seroprevalence surveillance may overestimate prevalence of infection if patients are repeatedly surveyed on an annual basis because antibody often persists for more than a year. Unlike case surveillance, seroprevalence does not distinguish between symptomatic and asymptomatic infection and it is symptomatic infection that better estimates the health burden of a disease.

#### *2.4. Ecological Studies*

#### Tick Vector and Mammalian Host Surveillance

Surveillance of tick vectors and/or reservoir hosts can provide a strong measure of risk of pathogen acquisition [3,70–78]. Detection methods include PCR, culture, and antibody testing. Tick vector or reservoir host surveillance only indirectly estimate the prevalence and location of human tick-borne infection but may provide a useful estimate of infection risk that complements results of human studies. In a comparative study of human and tick surveillance, incidence of Lyme disease and babesiosis were determined by reports of physicians to the Connecticut and Massachusetts Departments of Health and by reports of selected research study physicians in private practice in northeastern Connecticut and Nantucket, Massachusetts. The results of the study suggest that tick-borne surveillance can provide an early warning system for the emergence of tick-borne emerging infections [70].

#### *2.5. Genomics*

Genomics is an interdisciplinary branch of molecular biology that consists of the study of the structure, function, evolution, mapping, and editing of genomes. It focuses on the characterization and quantification of all the genes and their interactions that affect the function of the organism. The study of genomics has provided important new insights into the genetic basis of pathogen populations, their structure, diversity, evolution, and emergence; as well as pathogenesis, biomarkers of detection, drug resistance markers, targets for novel therapeutics, and vaccines [37,44,79–82].

#### *2.6. Mathematical Modeling*

Mathematical modeling is an epidemiologic tool used to study population dynamics and infectious disease transmission [83,84]. Modeling has increasingly been recognized as an important technique used to inform disease prevention and control efforts. Models may range from simple to highly complex, containing any number of parameters and variables depending on the outcome under investigation and data availability. Garner and Hamilton describe the different categories of epidemiologic models, which are classified on the basis of "treatment of variability, chance and uncertainty (deterministic or stochastic), time (continuous or discrete intervals), space (non-spatial or spatial), and the structure of the population (homogeneous or heterogeneous)" [84]. For example, in one study, laboratory and field data were integrated into a mathematical model to determine whether host coinfection with *Borrelia burgdorferi* (the agent of Lyme disease) and *B. microti* significantly increases the likelihood of *B. microti* establishment in a new previously uninfected region [45]. In another study, it was found that a model predicted that tick-borne diseases spread in a diffusion-like manner in the northeastern United States with occasional long-distance dispersal and that babesiosis spread exhibits strong dependence on Lyme disease [41].

#### **3. Modes of Transmission and Demographics of Human Babesiosis**

*Babesia* spp. perpetuate in nature through a tick-vector and mammalian-host cycle [39]. Vectors and hosts differ for each species of *Babesia* and vary geographically but the basic tick–host transmission cycle is similar for all [1]. The life cycle for *B. microti* is shown in Figure 2 with *I. scapularis* as the tick vector but other tick species serve as vectors for other *Babesia* spp. (Table 1). *Peromyscus leucopus* is the primary reservoir for *B. microti* but other small mammals, such as shrews and chipmunks, can also serve as reservoir hosts for *B. microti* and other *Babesia* spp. [39,85]. Similarly, deer and other large mammals are favored hosts for adult ixodid ticks. In some *Babesia* spp., such as *B. divergens*, this transstadial transmission is supplemented by transovarial transmission from mother to egg [7,86]. Deer markedly amplify tick numbers and are largely responsible for the emergence of *Babesia* and other tick-borne infections over the last three decades in the Northeast and northern Midwest regions of the United States [39,70].

**Figure 2.** Transmission of *Babesia microti* and stages in the *Ixodes scapularis* tick vector life cycle. Female *I. scapularis* lay 2000–3000 eggs in the spring that hatch in early summer and produce larvae. Larval *I. scapularis* ticks become infected with *B. microti* when they take a blood meal from infected white-footed mice (*Peromyscus leucopus*) or other small rodent hosts in late summer. Fed larvae molt into nymphs and overwinter. During the following late spring, summer, and early autumn, infected nymphs transmit *B. microti* to uninfected mice or humans when they take a blood meal. In the autumn, nymphs molt into adults. Adult males and females preferentially feed and procreate on white-tailed deer (*Odocoileus virginianus*) but rarely on humans. The blood meal provides sufficient protein for female ticks to lay eggs. The tick life cycle is repeated when a new generation of larvae hatch from the eggs in the early spring to complete the tick life cycle. Deer do not become infected with *B. microti*. The inset panels from left to right show a *B. microti* ring form with a non-staining vacuole surrounded by cytoplasm (blue) and two small nuclei (purple), an amoeboid form, a tetrad form (also referred to as a Maltese cross), and an extracellular form (adapted from The New England Journal of Medicine, Edouard Vannier, and Peter J. Krause, Human Babesiosis, 2012, 366, 2397. Copyright (2021) Massachusetts Medical Society. Reprinted with permission [1]).

> *B. microti* are primarily transmitted by *I. scapularis* ticks and rarely through blood transfusion, organ donation, and transplacentally [1,39,87–89]. Babesiosis has been one of the leading causes of transfusion transmitted infection in the United States [87,90]. More than 250 cases have been reported and approximately one-fifth of these cases have been fatal [87]. Blood donor screening for *B. microti* is an effective preventative measure [91,92]. In 2020, the United States Food and Drug Administration recommended donor screening in 14 *B. microti* endemic states and Washington D.C. using approved PCR technologies. Initial data indicate that the numbers of transfusion-transmitted cases has markedly decreased.

> Ten cases of congenital babesiosis due to *B. microti* have been described [88,93]. Strong supportive evidence indicates that these cases were not due to transfusion or tick transmission and definitive evidence was available for several cases. Congenital babesia infection is not always severe in neonates and there have been no fatalities. *B. microti* infection also

has been reported in two kidney transplant recipients who received kidneys from a single infected kidney donor [89].

The peak age of reported human *B. microti* cases in the United States is between 60 and 70 years of age (Figure 3). Very few cases are reported in children. In contrast, serosurveys show that children are infected as frequently as adults. Children have much milder disease and the diagnosis is more often missed in children. Indeed, about 40% of children are asymptomatically infected compared with about 20% of adults [60,94]. Babesiosis is reported more frequently in males than females, presumably because they are more often exposed to tick-infested areas. Lawn maintenance workers and those with occupational exposure to ticks are at greater risk of tick-borne diseases than the general population.

**Figure 3.** Babesiosis cases by age in the United States. Babesiosis cases reported by age to the Centers for Disease Control and Prevention, United States between 2011 and 2018 are shown. The low numbers of cases in children is due to the mild clinical symptoms resulting from *B. microti* infection rather than exposure to the infection. Almost half of children are asymptomatically infected compared to about a fifth of adults. Thus, *B. microti*-infected children are not diagnosed as frequently as adults (adapted from the Centers for Disease Control and Prevention. Notifiable Diseases and Mortality Tables. MMWR Morb Mortal Wkly Rep 2016, 65(3) [95]).

#### **4. Human Babesiosis in the Americas**

#### *4.1. Overview*

The first case of babesiosis in the United States was described in 1968 in a California resident, although the species was not identified [32]. Two years later, a case of *B. microti* was described in Nantucket, Massachusetts [40]. Subsequent reports on Nantucket established this island as the first babesiosis endemic site. The disease became known as Nantucket fever [65]. Subsequently, cases were reported on Cape Cod, Massachusetts, and the New England mainland. The reports of babesiosis subsequently broadened from southern New England to include endemic areas from Delaware to Maine [41,42,44,81,82,96,97]. Recent genomic studies have established that the initial source of *B. microti* was not from Nantucket but rather from the mainland in southeastern New England [44,81,82]. A similar emergence of babesiosis in Wisconsin and Minnesota is ongoing [81,98].

The emergence of babesiosis in the Northeast is thought to be due to several factors, including increased recognition of babesiosis by health care workers and the general public, an increase in the human population, construction of homes near wooded areas where ticks

abound, and a marked increase in the deer population [41,71,96,97,99–101]. In the late 19th century, the number of deer in the United States had decreased to an estimated 300,000 due to hunting and the loss of forest habitat for farmland. Deer sightings in New England at that time were mentioned in local newspapers [39]. As farming moved to the Midwest and hunting declined, the deer population steadily increased to about 30 million in 2017. An increase in the white-tailed deer population has been accompanied by a marked increase in the *I. scapularis* population and a concomitant increase in the number of cases of Lyme disease, while removal of deer from specific locations has greatly diminished the number of ticks and cases of Lyme disease [39,102–104]. Interestingly, Lyme disease has spread more widely than babesiosis, in part because *B. microti* is less efficiently transmitted than *B. burgdorferi* [41,45]. There are large areas of the Northeast and northern Midwest where Lyme disease is endemic but babesiosis is not. There are no areas where babesiosis is reported in the absence of Lyme disease (Figure 4). Laboratory studies suggest that Lyme disease/babesiosis coinfection enhances the transmission of babesiosis and it has been hypothesized that the establishment of Lyme disease in an area is a prerequisite for the establishment of babesiosis [43,45]. Furthermore, birds can serve as hosts for *B. burgdorferi* but not *B. microti*. Larval ticks may attach and feed on *B. burgdorferi*-infected birds and be deposited hundreds of miles away where they can then establish a new site of infection. Both *B. burgdorferi* and *B. microti* can spread from one infected colony of mice to an adjacent colony but spread in this case is much slower than with birds [39,41].

**Figure 4.** Human babesiosis occurs within Lyme disease endemic areas in the United States. Lyme disease and human babesiosis have been nationally notifiable conditions since 1991 and 2011, respectively. The names of counties that reported cases of Lyme disease and/or babesiosis from 2011 to 2013 were obtained from the Centers for Disease Control and Prevention. Counties with three or more cases of Lyme disease but fewer than three cases of babesiosis are depicted in green. Counties with three or more cases of Lyme disease and three or more cases of babesiosis are depicted in gray. No county reported three or more cases of babesiosis but fewer than three cases of Lyme disease (adapted from Diuk-Wasser M, Vannier E, Krause PJ. Coinfection by *Ixodes* tick-borne pathogens: Ecological, epidemiological, and clinical consequences. Trends Parasitol 2016, 32, 30–42 [43]).

#### *4.2. United States*

#### 4.2.1. *Babesia microti* Infection

Currently, 14 states account for the vast majority of babesiosis cases in the United States and most are due to *B. microti*. These states include Connecticut, Delaware, Maine, Maryland, Massachusetts, Minnesota, New Hampshire, New Jersey, New York, Pennsylvania, Rhode Island, Virginia, Vermont, and Wisconsin [46,95]. Geographic modeling suggests that babesiosis will continue to emerge in the United States. The areas presently endemic for babesiosis and Lyme disease are expanding toward each other from the Northeast and Midwest. It has been postulated that a continuous endemic band of these two diseases may someday extend from Minnesota to the East coast. Lyme disease also is expanding into southeastern Canada and this is thought to be due, at least in part, to climate change [105,106].

Clinical manifestations of *B. microti* illness vary from subclinical illness to fulminating disease resulting in death [1,39,60,100,107]. Fever typically develops after a gradual onset of malaise, anorexia, and fatigue and may reach 40 ◦C (104 ◦F) [1,96,100,108]. Other common symptoms include chills, sweats, myalgia, arthralgia, nausea, and vomiting. Physical examination of *B. microti*-infected patients reveals fever and occasionally mild splenomegaly, hepatomegaly, or both. Abnormal laboratory findings include hemolytic anemia, elevated renal function and liver enzyme levels, and thrombocytopenia [58,96, 100]. The illness usually lasts for a week or two but occasionally several months, with prolonged recovery taking up to 18 months [58,109]. Persistent parasitemia and clinical and microbiological relapse have been described for as long as 27 months after the initial episode, due in part to the development of antibiotic resistance [58,81,110–112]. Severe *B. microti* illness requiring hospital admission is common in patients with splenectomy, malignancy, HIV infection, hemoglobinopathy, chronic heart, lung, or liver disease, organ transplantation, acquisition of babesiosis through blood transfusion, and in newborn infants and the elderly [1,2,4]. Complications include severe hemolytic anemia, congestive heart failure, acute respiratory distress syndrome, disseminated intravascular coagulopathy (DIC), renal failure, coma, and shock [54,100,107].

#### 4.2.2. *Babesia duncani* Infection

In 1991, a 41-year-old resident of Washington State presented with viral-like symptoms and was diagnosed with babesiosis. The causative pathogen was propagated in hamsters and was found to be morphologically similar but genetically and antigenically distinct from *B. microti* [34]. The organism was named WA-1. Eight additional cases of babesiosis with recovery of the same causative *Babesia* pathogen were subsequently reported in California and Washington states. The *Babesia* were found to be morphologically, ultrastructurally, and genetically indistinguishable from one another and were subsequently named *Babesia duncani* [48]. Two additional cases have been described in California and Oregon, respectively. The primary vector is *Dermacentor albipictus* [113]. Limited data suggests that the clinical manifestations of these cases are similar to those of *B. microti*. There is a marked difference in disease severity in hamster and C3H/Hen mouse models, however, as *B. duncani* causes fatal illness while *B. microti* causes mild or asymptomatic infection [114,115].

#### 4.2.3. *Babesia divergens*-Like Infection

In 1996, Herwaldt and colleagues described a fatal case of babesiosis in a 73-year-old asplenic resident of Missouri who was infected with a *Babesia* that shared morphologic, antigenic, and genetic characteristics with *B. divergens*. The patient had previous exposure to cattle. The pathogen was named MO1 [36]. Four similar cases of *B. divergens*-like organisms have subsequently been described, none with exposure to cattle: (i) a 56 year old asplenic male resident of Kentucky who survived [49]; (ii) an 82-year-old asplenic male resident of Washington State with hypertension and secondary renal insufficiency who survived [50]; (iii) an 81-year-old asplenic Arkansas resident with diabetes, coronary artery disease, chronic obstructive pulmonary disease, a history of mitral valve replacement,

hypertension, and GI bleeding, who died [51], and (iv) a 60-year old asplenic female resident of Michigan who developed multiple organ failure but survived [52]. These cases were similar to those of *B. divergens* cases from Europe, where almost all have occurred in asplenic individuals and many have died (see below).

#### 4.2.4. Coinfection

Several different human pathogens cycle between *I. scapularis* ticks and mammalian reservoir hosts in the United States, including *Anaplasma phagocytophilum*, *Babesia microti*, *Borrelia burgdorferi*, *Borrelia mayonii*, *Borrelia miyamotoi*, deer tick virus (Powassan virus), and *Ehrlichia muris*-like organism [116]. These pathogens differ in their geographic range within the Northeast and northern Midwest. In areas where two or more pathogens are enzootic, simultaneous infection (coinfection) may occur. In the first case series of coinfection, the frequency and clinical outcome of Lyme disease and babesiosis alone were compared with those of Lyme disease and babesiosis coinfection [47]. Eleven percent of Lyme disease patients experienced coinfection while 72% of babesiosis patients had coinfection. This was expected because of the much larger number of Lyme disease patients compared with babesiosis patients. Lyme disease patients had a greater number of symptoms for longer duration if they were coinfected with *B. microti* [43,47]. The percentage of patients experiencing coinfection varies geographically and depends on the relative incidence of Lyme disease and babesiosis.

In addition to exacerbating human disease severity, *B. microti-B. burgdorferi* coinfection appears to increase *Babesia* parasitemia in the natural mouse reservoir, leading to greater transmission of *B. microti* from mouse reservoir to tick vector [45]. This enhancement of otherwise less transmissible *B. microti* may help explain why babesiosis has emerged more slowly than *B. burgdorferi* and is only found in areas of the United States where Lyme disease is endemic. Additional data suggests that coinfection provides a survival advantage for both *B. microti* and *B. burgdorferi* [43].

#### *4.3. Canada*

The first case of babesiosis in a Canadian resident was reported in 1999 [117]. The patient had traveled to Nantucket, Massachusetts six weeks prior to disease onset, indicating that the *Babesia* sp. identified on blood smear may not have been acquired indigenously. A second case of babesiosis was reported in 2001 in a 53-year-old Canadian resident who most likely acquired infection through blood transfusion from an asymptomatic *B. microti* positive donor [16]. The donor was thought to have acquired his infection in Cape Cod, Massachusetts. The first definitive case of locally acquired babesiosis in Canada was reported in a seven-year-old asplenic resident of Manitoba [17]. The child had not traveled outside Manitoba and never had a blood transfusion. *Babesia* were demonstrated on blood smear and *B. microti* was identified as the causative *Babesia* sp. by PCR. *I. scapularis* ticks infected with *B. microti* have been found in six different localities in Manitoba. Recently, two cases of *Babesia odocoilei* have been described with typical symptoms of babesiosis and positive PCR testing [118].

#### *4.4. Mexico*

A *Babesia* serosurvey was performed in Las Margaritas, Mexico in 1976. The sera of one third of 101 study subjects reacted against a dog *Babesia* antigen (*Babesia canis*) [59]. Three seropositive residents were found to be infected with *Babesia* when their blood was injected into splenectomized hamsters and *Babesia* were isolated from the hamsters. The *Babesia* species could not be identified. Four decades later, babesiosis due to *B. microti* was described in Yucatan State, Mexico [27]. The four patients ranged in age from 8 to 14 and lived in close proximity to each other in a rural area of eastern Yucatan. All subjects had tick bites or lived in tick-infested areas. All experienced mild to moderate illness with fever and three also experienced fatigue, arthralgia, and myalgia. The diagnosis was confirmed and the infecting species identified by amplification of *B. microti* DNA using PCR. All were

given chloroquine and had a full recovery despite the fact that chloroquine is not effective for the treatment of human babesiosis.

#### *4.5. South America*

Two cases suggestive of babesiosis were reported in 2003 in South America. One was a 37-year-old resident of Puerto Berrio, Colombia who had fever, chills, sweats, weakness, and bone aches. *Babesia* parasites were identified on thin blood smear. A PCR was not performed but the patient had an antibody titer of 1:64 against *Babesia bovis* antigen [19]. The second case was an asymptomatic 2-year-old from Brazil with hepatoblastoma who had a positive blood smear for *Babesia* [15]. No *Babesia* PCR or antibody testing were performed.

In a survey of 300 residents of two rural towns (Turbo and Necocli) in Colombia where cattle ranching is an important industry, four subjects tested positive for *B. bovis* by PCR, including two who were blood smear positive [119]. Another two residents tested positive for *B. bigemina* by PCR, including one whose blood smear was positive. Three of these subjects were symptomatic with fever and/or headache and three were asymptomatic. Human babesiosis due to *B. bovis* and *B. bigemina* had not previously been described.

Nine cases of asymptomatic *B. microti* infection were discovered among 271 healthy residents of two rural towns in southeastern Bolivia [14]. All nine cases had *Babesia* identified on thin blood smear and further characterized as *B. microti* by PCR and molecular sequencing. All cases were seropositive when tested with a standard *B. microti* immunofluorescence antibody (IFA) assay.

A 72-year-old patient from Ecuador with chronic abdominal pain moved to Chicago and two months later developed fever, chills, headaches, myalgia, dry cough, nausea, vomiting, and diarrhea. He was admitted to the hospital and diagnosed with malaria based on his country of origin, symptoms, a positive blood smear showing intraerythrocytic ring forms (parasitemia 0.5%), and positive *P. falciparum* IgG antibody. A blood sample sent to the CDC was positive for *B. microti* by PCR. His infection resolved on atovaquone and proguanil [20].

In summary, there is evidence of human *B. microti* and other *Babesia* spp. infection in South America. Additional studies are necessary to better define the scope of the problem there, including confirmation of other *Babesia* species causing human infection.

#### **5. Human Babesiosis in Europe**

#### *5.1. Overview*

The first documented case of human babesiosis anywhere in the world was reported in the former Yugoslavia in 1957 [33]. The affected patient was a splenectomized farmer who succumbed to severe hemolytic anemia. The parasite species was never determined but *B. bovis* was found in the cattle he tended [120]. Since then, more than 50 cases of babesiosis have been reported on the European continent [1,5,121–123]. The predominant pathogen in Europe is *B. divergens,* however, *B. microti* and *B. venatorum* have been identified in a small number of cases [35,124,125]. A case of *B. divergens*-like infection has been reported in the Canary Islands (Spain) [8]. A comprehensive review of human babesiosis in Europe by Hildebrandt et al. (2021) documented a total of 51 autochthonous cases, with 35 attributed to *B. divergens*, 11 to *B. microti*, and 5 to *B. venatorum* [2]. Epidemiologic surveys have indicated widespread distribution of *B. divergens* and its associated tick vector, *Ixodes ricinus,* throughout Europe [126]. Recent seroprevalence reports suggest a much higher clinical incidence than has been described in the extant literature to date [127]. Quantitation of true babesiosis incidence across Europe remains a challenge because symptoms often manifest non-specifically, immunocompetent individuals are frequently asymptomatic, and babesiosis is not a notifiable disease in many countries [128].

#### *5.2. Babesia divergens*

*B. divergens* is the primary causative agent of human babesiosis in Europe and is endemic in the European cattle population. Gray (2006) described the ecological landscapes of countries with the highest incidence of bovine babesiosis as having significant tick populations in "rough open hill-land or damp low-lying meadows" and "where woodland frequently abuts cattle pasture" [129]. Over half of the cases of European babesiosis have been reported in France and the British Isles, with at least 10 other countries represented in single case reports [31,122,130–133]. Prevalence of babesiosis is reportedly increasing, and the European Center for Disease Prevention and Control have identified several factors driving this trend: landscape modifications affecting tick populations, deer population growth, human activity in infested areas, and dissemination of pathogens through cattle movement (https://www.ecdc.europa.eu/en/all-topics-z/babesiosis/facts-about-babesiosis, accessed on 27 July 2021). Disease emergence at increasingly northern latitudes in Europe has been recently observed. Mysterud and colleagues analyzed longitudinal tickborne disease incidence data from Norway and found that this emergence is linked to tick vector distribution [134]. *I. ricinus* is the primary vector of *B. divergens* and is widely distributed across Europe [135]. Primary host species include domesticated cattle, [126], roe deer, and other cervids (e.g., moose, red deer, reindeer, sika deer) [136].

*B. divergens* infections are characterized by fulminant disease and all but a few cases have been reported in asplenic patients [5,123,128]. Factors that predispose patients to severe disease include the extremes of age and other causes of immunocompromised clinical status [122,137]. After an incubation period of 1–3 weeks, *B. divergens* symptoms generally have a rapid progression with high fever, chills, sweats, headache, myalgia, hemolytic anemia, and hemoglobinuria [5]. Mortality associated with *B. divergens* infection, often due to multiorgan failure, was previously estimated to be as high as 42% but is improving. Better outcomes are thought to result primarily from more aggressive therapy, including intravenous antibiotics and the early use of exchange transfusion [5]. Two recent publications have challenged this "classic description of babesiosis in Europe." Martinot et al. described two exceptional cases of severe babesiosis in healthy, young, immunocompetent patients in France, and Gonzalez described a similar case in Spain [123,138].

#### *5.3. Babesia venatorum*

*B. venatorum* is an emerging public health concern in Europe due to its widespread zoonotic presence [136]. *B. venatorum*, formerly referred to as *Babesia* sp. EU1, is closely related to *B. divergens* and *B. odocoilei* [35,139]. Wild hosts include roe deer and moose [136]. The parasite has also been detected in captive reindeer and domesticated sheep [75,140–142]. The *I. ricinus* tick acts as both vector and reservoir. Cases in humans have thus far been reported in Austria, Germany, and Italy [35,124]. Case reports have described disease manifestations ranging from mild to moderately severe, which resolve with antimicrobial therapy, even in the setting of asplenia and lymphoma. The clinical presentation of *B. venatorum* infection is generally less severe compared to that of *B. divergens* [5].

#### *5.4. Babesia microti*

Cases of *B. microti* infection have been reported from Austria, Germany, Italy, Poland, Spain, and Switzerland [35,125,143–146]. The first evidence of human *B. microti* infection in Europe was a report of seropositive residents in Switzerland in 2002 [147]. A number of serosurveys have shown a wide range of *B. microti* seropositivity depending on the location and study population (e.g., general public, forest workers, Lyme disease coinfected subjects). *B. microti* seropositivity has ranged from 0.5% to 32% in study populations in Belgium, France, Germany, Italy, Poland, Sweden, and Switzerland [139,147–153] Furthermore, Hunfeld et al. (2002) reported that IgG seroprevalence rates were higher for *B. microti* (9.3%) than for *B. divergens* (4.9%) among patients exposed to ticks in Germany. [139] These seroprevalence data indicate that there is more human *B. microti* infection in Europe than currently identified.

#### *5.5. Babesia crassa-Like Agent*

*Babesia crassa* is a relatively uncommon *Babesia* species with documented infection in sheep in Iran and Turkey [154]. A single case of *B. crassa*-like infection has been reported in Europe in Slovenia [155]. The patient in question was asplenic and recovered after standard antibiotic treatment. Cases subsequently have been described in China.

#### **6. Babesiosis in Asia**

#### *6.1. Overview*

Several countries in Asia have reported human cases of babesiosis, including China, India, Japan, Korea, and Mongolia. In addition to previously documented human *Babesia* pathogens, several new *Babesia* species have been found to infect humans. As with any single case report of a novel *Babesia* species or report of a known *Babesia* sp. in a new region, identification of additional cases and pathogen isolation from local tick vectors and mammalian hosts will help confirm original findings [22,156]. The increasing interest and reports of human babesiosis in Asia are likely to reveal additional species and new areas of endemicity.

#### *6.2. China*

#### 6.2.1. Human Infection

Outside the United States, the greatest number of human babesiosis cases are reported in China. China is the only country, other than the United States, where babesiosis has been shown to be endemic. Babesiosis in China is considered an emerging public health threat [3,6,157,158]. Among the human *Babesia* spp. identified to date, four (*B. microti*, *B. divergens*, *B*. *venatorum*, and *B. crassa*-like agent) have been confirmed to cause human infections in China [11,35,159–165]. Studies in western China more than a decade before the first official report of human babesiosis in Yugoslavia described *Babesia*-like intraerythrocytic organisms associated with febrile illness that may have been *Babesia* [6].

#### 6.2.2. *Babesia venatorum*

*B*. *venatorum* was found to cause infection over a two year study period in Heilongjiang province in northeastern China, indicating endemic transmission. The majority of tickborne cases in China are found in this province. Jiang et al. screened 2912 individuals for microscopic, PCR, or animal inoculation evidence of *Babesia* spp. infection in patients who reported a recent tick-bite and who sought hospital care between 2011 and 2014. Results showed that 48 (0.16%) of these patients had *B. venatorum* infection [12]. The *B. venatorum* 18S RNA gene sequences from all 48 patients were identical and differed from European *B. venatorum* parasite isolates by only two nucleotides. These data suggested a common origin of *B. venatorum* spp. in parasites circulating in northeastern China and Europe. Only five cases of *B. venatorum* had previously been identified, four of which were in Europe and one in a child in China [6,35,124,164,166,167].

#### 6.2.3. *Babesia crassa*-Like Agent

A similar study led to the discovery of *B. crassa*-like pathogen as another causative agent of endemic human babesiosis in China. Between May 2015 and July 2016, Jia et al. screened 1125 residents of Heilongjiang Province for evidence of *Babesia* spp. infection who experienced fever and recent tick-bites. Of these participants, 5.0% (58/1125) demonstrated the presence of a novel *B. crassa*-like species in their blood, based on species-specific PCR testing and nucleotide sequencing [11]. *B. crassa*-like parasites were visualized on thin blood smears and showed ring, ameboid (<3 µm in size), and tetrad forms. The authors characterized the severity of disease manifestations as mild to moderate. Interestingly, 7.5% of healthy, asymptomatic residents of the area tested positive for *B. crassa*-like infection, suggesting that many human babesiosis cases due to *B. crassa*-like pathogen go undetected in China [11].

DNA samples also were collected from 1732 adult ticks from May to July 2014 from the same study area. Nine *I. persulcatus* and *Haemaphysalis concinna* ticks showed the presence of *B. crassa*-like species. Blood samples collected from 5 of 1125 sheep contained *B. crassa*like DNA [11]. The *B. crassa*-like species is phylogenetically related to *B. crassa*, a large *Babesia* parasite of sheep in Turkey and Iran [154,168]. The near full length *B. crassa*-like 18S rRNA gene sequences showed 96.7% and 97.7% sequence similarities with the *B. crassa* sequences, respectively, from sheep in those countries [11].

#### 6.2.4. *Babesia microti*

*B. microti* is another important *Babesia* sp. that causes human babesiosis in China [158]. Phylogenetic analyses based on the sequences from the 18S rRNA gene have revealed that *B. microti* from China are phylogenetically similar to those from Japan and Switzerland [6]. Clinical cases attributed to *B. microti* have been reported sporadically from Zhejiang, Yunnan, and Guangxi provinces [158,160]. Accurate diagnosis of clinical babesiosis is a challenge where *B. microti* babesiosis and malaria coexist in the same area in southwestern China, specifically Yunnan Province along the China–Myanmar border. The first reported cases of co-infections of *B. microti* and *Plasmodium* spp. were discovered there in 2012– 2013 [165]. *B. microti*, *P. falciparum*, *P. vivax*, and *P. malariae* infections were identified among 449 febrile patients. Eight patients (1.8%) had infection with *B. microti* alone while 10 (2.2%) were co-infected with *B. microti* and either *P. falciparum* or *P. vivax* [165]. These results clearly illustrate a possible hidden clinical burden of *B. microti* in malaria endemic areas where babesiosis is not known to exist. Furthermore, patients experiencing febrile illness with intraerythrocytic parasites on blood smear may be misdiagnosed as having malaria when they actually have babesiosis.

*B. microti* has been shown to be transmitted by blood transfusion in the United States and Japan. Very limited data is available on the transmission risk of *B. microti* in Chinese blood donors. A single case of transfusion-associated babesiosis in China has been reported. *B. microti* was identified as the causative agent [169]. Large scale molecular and serological surveys to assess *Babesia* spp. risk among random blood donors in China are not yet available. A 2016 pilot serosurvey of blood donors in Heilongjiang Province revealed that 13 of 1000 (1.3%) donors had antibodies against *B. microti* parasites by the immunofluorescence antibody assay [161]. This *B. microti* antibody positivity rate is comparable to rates observed in blood donors in endemic areas in the northeastern United States [170]. These results provide further evidence that the prevalence of *B. microti* transmission in China may be significantly higher than currently realized and might be comparable to prevalence in the United States.

#### 6.2.5. *Babesia divergens*

In recent years, laboratory screenings of probable cases of babesiosis in patients presenting to Chinese hospitals with recent tick bites have yielded surprising findings that are suggestive of the presence of novel *Babesia* spp. The first case of *B. divergens* (cattle *Babesia* sp.) infection in China was identified in a patient in 2011. The 18S rRNA gene sequence from this individual had 98.4% similarity with the gene of *B. divergens* in Switzerland [162]. A subsequent study in Gansu Province of 754 patients who visited a hospital for a tick bite between April and March 2016 showed that 10 patients (1.3%) had *B. divergens* infections, based on positive PCR tests [163]. *B. divergens* sequences from this study site were 99.9% identical to sequences of *B. divergens* from Europe. Interestingly, *B. divergens* infection has never been identified in cattle in China, possibly indicating a different reservoir host for this *Babesia* sp. Another salient feature of this study was that all 10 *B. divergens* infected patients were immunocompetent and only two had clinical symptoms at the time of sample collection.

#### 6.2.6. Tick-Vectors and Animal Hosts of *Babesia* spp. in China

Several entomological and molecular studies have allowed quantitation of tick-vector and reservoir host infection rates, as well as geographic distribution of *Babesia* spp. in China. Fang et al. (2015) published a comprehensive overview of tick-borne infections in tick vectors, animal hosts, and humans [3]. The authors reported a total of 33 emerging tick-borne agents that have been identified in mainland China, including 11 species of *Babesia*. Their analyses showed that transmission of *Babesia* spp. is associated with 13 tick species. Although more prevalent in the northeastern regions, *Babesia* spp. were distributed throughout China.

Among *Babesia* spp. that infect humans, *B. venatorum* has been reported in *I. persulcatus* ticks from northeastern China [3]. *B. crassa*-like agent has been detected in *I. persulcatus* and *H. concinna* ticks from sheep in the same area in Heilngjiang Province [11]. *B. microti* has been identified over a broad expanse of China, including, (i) *I. persulcatus* and *H. concinna* ticks and striped field mice and reed voles in Heilongjiang Province, (ii) *H. longicornis* ticks on dogs from Henan Province, and (iii) rodents from Fujian, Zhejiang, Henan, and Heliongjiang provinces. *B. divergens* has been detected in *I. persculcatus*, *H. concinna,* and *Haemaphysalis japonica* ticks and in striped field mice in Heilongjiang Province. *Babesia* spp. that have not been shown to cause human infection in China include *B. ovis*, *B. major*, *B. ovata*, *B. orientalis*, *B. motasi*, *B. caballi*, *Babesia* sp. Kashi, and *Babesia* sp. Xinjiang [3]. More recently, Xia et al. have performed genotyping of *Babesia* spp. in a total of 2380 *I. persulcatus* and *H. concinna* ticks in a narrow forested area at 30 sampling points in northeastern China based on the 18S rRNA gene sequences [76]. Results showed that 23 (0.97%) of *I. persulcatus* ticks tested positive for five *Babesia* spp.—*B. bigemina*, *B. divergens*, *B. microti*, *B. venatorum* and one novel strain HLJ-80. Thirteen *H. concinna* ticks were positive for the following *Babesia* spp.—*B. bigemina*, *B. divergens*, three genetic variant forms of HLJ-874, and eight other *Babesia* variants represented by HLJ 242, which were similar to *B. crassa* [76]. The authors concluded that each site contained 5–6 different *Babesia* spp., several of which are capable of infecting humans. Additionally, Kobi-type and Otsu-type *B. microti* have been detected in wild rodents in Yunnan Province [171]. Overall, the presence of a number of *Babesia* spp. and their genetic variants infecting tick vectors and animal hosts indicate a high *Babesia* transmission risk to humans living in different parts of China.

#### *6.3. India*

A single case of human babesiosis was described in a resident of north central India in 2005. The diagnosis was confirmed by identification of *Babesia* on thin blood smear but the species was not identified. Antigen tests for *Plasmodia* were negative [22,23].

#### *6.4. Japan*

In 1980, Shiota et al. documented the presence of *B. microti* parasites in blood films collected from Japanese field mice [172]. The only autochthonous case of human babesiosis that has been reported from Japan was in a patient who acquired infection through blood transfusion during admission to Kobi University Hospital, Hyogo Prefecture in 1999 [24]. *B. microti* parasites were confirmed by blood smear microscopy and PCR analysis. The parasite isolated from the index patient's blood sample and from a blood sample inoculated and propagated in SCID mice were identified as a *B. microti*-like parasite, which had a 99.2% sequence homology with the *B. microti* reference strain from the US [24]. Although a blood sample from the implicated asymptomatic donor collected eight months after the index donation was negative for *B. microti* parasites by blood smear microscopy and PCR analysis, inoculation into SCID mice allowed detection of *B. microti* parasites that had sequence identity with the parasite isolate from the blood recipient [173]. *B. microti*parasites exhibiting a similar genotype as the index patient and the asymptomatic blood donor were also isolated from a field mouse near the donor's residence, indicating enzootic and zoonotic transmission of *B. microti* in the area [173].

Molecular surveillance studies in the presumed *I. persulcatus* tick vector and field mouse reservoir host have demonstrated the presence of *Babesia* spp. throughout Japan with a potential for human transmission [173]. A field survey in Hokkaido Prefecture revealed the presence of *B. divergens* (Asia lineage) parasites in *I. persulcatus.* The presence of *B. microti* (United States lineage) and *B. venatorum* (strain Et65) were also noted in the same tick species [174]. Sika deer (*Cervus nippon*) were shown to carry *B. divergens* parasites in different Japanese prefectures [175]. In a more recent study, hard ticks belonging to the genera Ixodes and Haemaphysalis collected from sika deer in Hokkaido were found to harbor DNA for *B. microti*, *B. microti* Hobetsu, and *B. divergens*-like (Bab-SD) parasites [176]. Together, these studies suggest a wide-spread presence of *Babesia* spp. in tick vectors, mouse reservoir hosts, and humans in Japan.

#### *6.5. Korea*

Two cases of human babesiosis have been documented in Korea. In the first case, a blood sample from a patient contained paired pyriform and ring forms of *Babesia* parasites. The parasite isolate was named *Babesia* sp. KO1 and was found to be genetically related to sheep *Babesia* in China [26]. In the second case, a parasite isolate from a symptomatic patient was found to be closely related to *B. motasi*, a sheep parasite. Tick samples collected nearby the patient's residence demonstrated the presence of *B. microti* and *B. motasi* DNA (98% homology) [25]. Limited data is available for the tick-vectors and reservoir hosts of *Babesia* spp. in Korea. In one study, *B. microti* parasites (United States type) were detected by PCR in blood samples from wild animals in Gangwon-do Province [177]. In another study, *B. microti* (United States type) DNA was detected in blood samples from *Apodemus agrarius* (striped field mouse) but was absent from the other small mammals that were screened [28].

#### *6.6. Mongolia*

A survey of 100 asymptomatic farmers in Selenge province, Mongolia revealed that 7% had *B. microti* antibody and 3% had amplifiable *B. microti* DNA in their blood [28]. In a more recent study, a third of 63 questing *I. persulcatus* ticks were found to be infected with *B. microti* (United States type) in Selenge province in Mongolia [178].

#### **7. Babesiosis in Africa**

There have been very few cases of human babesiosis reported on the African continent to date. Human babesiosis caused by unknown species have been described in Egypt and Mozambique [8,21,121]. Two cases of babesiosis due to unknown species were reported in South Africa [29]. A 2018 case study described by Arsuaga et al. illustrates the difficulties of diagnosing babesiosis in the malaria-endemic areas of Cameroon and subsequently, Equatorial Guinea [179]. The complicated travel history of the patient in question coupled with the lack of available surveillance data on ticks and vertebrate reservoirs of *Babesia* species rendered it impossible for the authors to determine the definitive source of infection. Bloch and colleagues attribute the dearth of reported cases in Africa to a lack of surveillance data and to clinical and diagnostic overlap of *Babesia* with *Plasmodium* spp. in endemic areas [161]. In a pilot seroprevalence study, these authors examined seroreactivity among children in the Kilosa district of Tanzania. They concluded that *Babesia* may be present in the area, but that the potential for serological cross-reactivity and false positivity between *Babesia* and *Plasmodium* spp. impedes definitive conclusions about seroprevalence [161].

#### **8. Babesiosis in Australia**

A single autochthonous case of human babesiosis has been documented in Australia. Blood smear microscopy and molecular analysis revealed *B. microti* (United States type) as the infecting parasite [13]. A single imported case of babesiosis caused by *B. microti* infection also has been reported [180]. No evidence of *B. microti*-specific antibodies in 7000 blood donors and 29 clinically suspected babesiosis patients was detected in a serosurvey at

multiple study sites in eastern Australia, leading the authors to conclude that transmission of *B. microti* is uncommon in this large region [181]. Babesiosis is a prevalent disease in cattle in Australia and is caused by *B. bigemina* and *B. bovis* [182]. Babesiosis is also prevalent in dogs where infecting species are *B. canis*, *B. vogeli*, and *B. gibsoni* [183]. Molecular studies demonstrating a tick-vector and reservoir-host for human *Babesia* spp. are lacking.

#### **9. Conclusions**

Human babesiosis is a worldwide emerging health problem that imposes a major disease burden, especially on the expanding older population and immunocompromised patients. Numerous studies indicate that the true number of *Babesia*-infected patients is markedly underestimated. As the infection continues to emerge, the number of affected individuals is likely to increase. Improved surveillance, as well as development of new antibiotics, supportive therapies, and a vaccine will all be important in limiting the impact of this disease.

**Funding:** We thank the Llura A. Gund Laboratory for Vector-borne Diseases and the Gordon and Llura Gund Foundation for financial support. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** We thank Molly Missonis for her assistance in writing this manuscript.

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

#### **References**


## *Review* **Ticks, Human Babesiosis and Climate Change**

**Jeremy S. Gray 1,\* and Nicholas H. Ogden 2,3**


**Abstract:** The effects of current and future global warming on the distribution and activity of the primary ixodid vectors of human babesiosis (caused by *Babesia divergens*, *B. venatorum* and *B. microti*) are discussed. There is clear evidence that the distributions of both *Ixodes ricinus*, the vector in Europe, and *I. scapularis* in North America have been impacted by the changing climate, with increasing temperatures resulting in the northwards expansion of tick populations and the occurrence of *I. ricinus* at higher altitudes. *Ixodes persulcatus*, which replaces *I. ricinus* in Eurasia and temperate Asia, is presumed to be the babesiosis vector in China and Japan, but this tick species has not yet been confirmed as the vector of either human or animal babesiosis. There is no definite evidence, as yet, of global warming having an effect on the occurrence of human babesiosis, but models suggest that it is only a matter of time before cases occur further north than they do at present.

**Keywords:** *Ixodes ricinus*; *Ixodes scapularis*; *Babesia microti*; *Babesia divergens*; climate; global warming

**Citation:** Gray, J.S.; Ogden, N.H. Ticks, Human Babesiosis and Climate Change. *Pathogens* **2021**, *10*, 1430. https://doi.org/10.3390/ pathogens10111430

Academic Editors: Estrella Montero, Cheryl Ann Lobo, Luis Miguel González and Lawrence S. Young

Received: 26 September 2021 Accepted: 1 November 2021 Published: 4 November 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

#### **1. Introduction**

According to the 6th IPPC report, published in August 2021, global temperatures over the next 20 years are expected to "reach or exceed an average of 1.5 ◦C, unless there are immediate, rapid and large-scale reductions in greenhouse gas emissions". Given that similar predictions were made, though with longer time scales, in each of the previous five reports, it now seems that such a global temperature increase is highly likely. This will result in an increasing number of heat waves, longer warm seasons and fundamental changes in rainfall patterns. Indeed, the first signs of these changes are already evident, most obviously in the natural world and relevant here in relation to arthropod vectors of disease [1]. It has been suggested that complex effects of climate change on both host communities and arthropod vectors could result in unanticipated spillover of pathogens from reservoir hosts into domesticated animals or humans resulting in disease emergence, depending on the host range of the pathogen [2], but the risk of emergence of novel *Babesia* spp. is unknown.

The risk of human babesiosis can be affected by climate change in at least three different ways. Firstly, as poikilothermic organisms, the ixodid tick vectors of human babesiosis and the babesia pathogens within them can respond directly to changes in ambient conditions; secondly and more indirectly, both ticks and the vertebrate reservoirs of the pathogens can be affected by the impact of climate change on vegetation, resulting in changes to habitats (e.g., beech woods [3]), and to host food sources (e.g., masting events [4,5]), thirdly anthropogenic responses to climate change, notably human behaviour, but also the management of livestock reservoirs of infection, will affect exposure to the vectors and therefore the risk of disease (Figure 1).

1

**Figure 1.** Factors determining the abundance and spread of *Ixodes* spp. Modified from Lindgren et al., 2000 [6].

The predominant vectors of human babesiosis are *Ixodes scapularis* transmitting *Babesia microti* in the USA, and *Ixodes ricinus*, transmitting *Babesia divergens* and *Babesia venatorum* in Europe [7]. *Babesia microti* also occurs in Europe, but human cases are extremely rare [8].

Several cases of *B. divergens* [9], *B. venatorum* [10] and *B. microti* [11], have been reported from China, and the vector, based on DNA detection, is suspected to be *Ixodes persulcatus* [11]. The same tick species is thought to be the vector of *B. microti* and an Asian lineage *B. divergens* in Japan [12,13]. Curiously, *I. persulcatus* has not been associated with either human or bovine babesiosis in Russia or Eastern Europe. Several other *Babesia* species in addition to *B. divergens*, *B. microti* and *B. venatorum* occasionally infect humans, but in most cases the identity of the vectors is unknown. The exceptions are *Babesia duncani*, which recent evidence suggests is transmitted by *Dermacentor albipictus* [14], a *Babesia crassa-*like parasite, probably transmitted by *I. persulcatus* or *Haemaphysalis concinna* [15], and an unnamed *Babesia* species in the USA, closely related to *B. divergens* and probably transmitted by *Ixodes dentatus*, a rabbit tick [16]. Since cases caused by these three *Babesia* species are rare, they will not be considered further here.

#### **2. Life Cycles and Ecology of the Human Babesiosis Vectors**

The three tick species responsible for most cases of human babesiosis, *Ixodes persulcatus*, *I. scapularis* and *I. ricinus* belong to the *Ixodes ricinus* species complex, consisting of at least another 15 species. They are three-host ticks, using separate hosts for each of the active stages, larva, nymph and adult, all of which engorge except for the male, which is probably not significantly involved in disease transmission. They are generalist species and feed on a very wide range of hosts, but there is some host selection, with larvae tending to feed preferentially on small mammals, nymphs on medium-sized mammals and birds, and adult females mainly on large mammals, such as deer and domestic livestock. However, there is a great deal of flexibility in these host preferences and large hosts can be heavily parasitised by the immature stages. Unlike most *Ixodes* species, which utilise hosts in nests and burrows, *I. persulcatus*, *I. ricinus* and *I. scapularis* attach to hosts in the open, using vegetation as ambush vantage points. When they have fed to repletion on their hosts over a few days, they drop off back into the vegetation, locate in the litter layer and commence

development to the next stage, or commence egg development and then oviposition in the case of the female.

The life cycles of all three tick species are characterised by distinct seasonal activity of questing ticks, partly regulated by ambient conditions, so that little or no questing behaviour occurs at very high or very low temperatures, however, diapause is also a significant regulating mechanism. Diapause can be defined as a form of hormonally controlled arrested development or delayed behaviour that occurs prior to seasonally unfavourable environmental conditions. Conditioning of the ticks results from entrainment by certain environmental stimuli, particularly day length, and usually lasts for a set period. Laboratory studies have shown that temperature can affect diapause directly [17], but temperature may be more important in determining rates of tick development in relation to the seasonally-determined diapause conditioning periods. The role of diapause in regulating the life cycles of *I. persulcatus*, *I. ricinus* and *I. scapularis* has been reviewed recently [18].

The three *Ixodes* vectors of human babesiosis have very wide distributions encompassing several climate zones, for example *I. ricinus* occurs from the western seaboard of Europe to as far eastwards as the Ural Mountains and from the Atlas Mountains in North Africa to Northern Norway, though it is scarce in arid regions of southern Europe. The range of *I. persulcatus* is even greater, extending from Eastern Europe to the temperate Far East, and *I. scapularis* occurs from the southern states of the USA through the eastern seaboard as far north as southern Canada. Despite such wide ranges, the distribution of these tick species is limited by their susceptibility to desiccation when off the host. During development and especially when host-seeking (questing) they are exposed to ambient conditions and therefore confined to habitats that include humid microclimates (>80% RH) at the base of the vegetation, where the ticks obtain water by secreting a hygroscopic fluid onto their mouthparts and then ingesting it. Since questing may continue for several weeks, the ticks must make several journeys from the surface vegetation to soil level to replenish their water supply. The drier the atmosphere the more such trips, all costing energy, so that in hot, dry conditions survival may be limited. *I. persulcatus* differs from the other two species in that the immature stages are more reluctant to climb the vegetation and tend to quest in the litter layer [19], and southern strains of *I. scapularis* show similar behaviour relative to those from more northern regions in the USA [20], which may be a heritable adaptation to the drier conditions in the south. The consequence of the requirement of these ticks for humid microclimates when off the host is that their typical habitats tend to be woodlands with a substantial layer of vegetation litter. Deciduous and mixed forests offer the most favourable conditions, but coniferous forests may also harbour substantial numbers of ticks. Additionally, open habitats of rough vegetation such as the sheep-grazed uplands of north-western Europe, where maritime climates maintain mild winters and high humidity due to frequent rainfall, can maintain large numbers of ticks [21].

Another factor determining distribution and survival is temperature, which affects both development and questing, the lower thresholds of which probably vary with species, with regional differences occurring within tick species [22]. Cold air temperatures seem to have a limited effect on actual survival. For example, *I. scapularis* placed at −20 ◦C in the laboratory die rapidly, but engorged ticks placed in the litter layer in suitable woodland habitats in Canada over the winter (where air temperatures can often fall to less than −30 ◦C) have daily mortality rates no greater than those in summer, probably due to the insulating capacity of the litter layer (reviewed in Ogden et al. [23]). Similarly, it has been observed in Germany that *I. ricinus* populations are adversely affected by air temperatures of less than −15 ◦C only when the insulating snow cover is absent [24]. In the context of global warming, high temperatures are obviously important as drivers of desiccation in limiting tick survival, but laboratory studies suggest that even in the presence of high humidity, *I. ricinus* may suffer much higher mortality when temperatures exceed 30 ◦C [25].

The third vital component ensuring establishment and survival of tick populations is the availability of adequate numbers of appropriate hosts. In most habitats of the *Ixodes* species considered here, deer are essential hosts for the maintenance of the tick populations, because they are the only animals that feed significant numbers of adult female ticks, although in agricultural settings *I. ricinus* is also maintained by livestock, especially sheep and cattle [21]. Large hosts can feed all tick stages, but in woodland habitats small mammals and birds are important hosts of the immature stages, and many are essential components of tick-borne diseases, such as Lyme borreliosis, tick-borne encephalitis, several rickettsioses and human babesiosis caused by *B. microti.*

It is notable that ticks are increasingly recorded in urban areas and in such settings hedgehogs (*Erinaceus* spp.), another host that can feed all tick stages and thus maintain small populations of *I. ricinus*, could theoretically maintain zoonotic *B. microti* in the absence of large hosts [26,27]. As yet there are no reports of such foci, partly no doubt because zoonotic *B. microti* genotypes are rare in Europe [8].

#### **3. The Roles of Reservoir Hosts of Human Babesiosis**

*Babesia microti* is considered to be the most important cause of human babesiosis since it is responsible for the vast majority of cases, particularly in the USA. However, a study published in 2003 by Goethert and Telford [28], revealed that this is not a single species but consists of a complex belonging to three distinct clades utilizing a wide range of hosts, mostly rodents, but also shrews, dogs, foxes and raccoons. In the USA, some bird species were implicated in a single study as reservoirs of a *B. microti-*like organism [29]. However, the genotype involved is not known, and the distribution pattern of endemic areas of *B. microti-*babesiosis in the USA does not support long-distance distribution of the pathogen by birds. At present there is no evidence for significant bird involvement in the transmission of zoonotic *B. microti* genotypes, but this topic needs further study. In the Goethert and Telford study [28], most of the zoonotic genotypes turned out to belong to a single clade prevalent in the USA (though not confined to that country) and often referred to as the US-type or *B. microti* sensu stricto (s.s.), found in woodland mice, shrews and chipmunks. In Europe, very few cases of human babesiosis have been described despite widespread infection of rodents [8] and transmission by *I. ricinus* [30]. Although these cases appear to have been caused by rodent parasites, their rarity suggests that zoonotic genotypes are uncommon in Europe. In many regions the parasite is transmitted by *Ixodes trianguliceps*, which rarely bites humans, further reducing the risk of zoonotic babesiosis [8].

*B. venatorum* is a relatively recently described European zoonotic species [31], which has since been reported to have caused many more cases in China [10]. In Europe, good evidence now exists that the reservoir host of *B. venatorum* is roe deer (*Capreolus capreolus*) [32,33]. Sika deer (*Cervus nippon*) probably fulfils this role in China. Thus, two zoonotic *Babesia* spp. (*B. microti* s.s. and *B. venatorum*) are firmly associated with woodland. The third species, *B. divergens*, has until recently been considered to be an exclusive cattle parasite. With the advent of molecular taxonomy this parasite has also been reported from red deer (*Cervus elaphus*) and roe deer (*C. capreolus*) [34], but there is no evidence for wild deer as a source of infection for cattle or vice versa, although splenectomised red and roe deer can evidently be infected with *B. divergens* from cattle [35]. Current data suggest that almost all isolates from human cases closely match bovine babesia sequences, only two with less than 99.9% 18S rRNA gene homology, and having little identity with babesia sequences from deer [8]. The host origins of these babesias are unknown. It must be concluded that at present there is no evidence for deer as a source of *B. divergens* infection of humans and that human cases are predominantly associated with cattle and thus with agricultural rather than woodland habitats.

#### **4. Expected Impacts of Climate Change on the Vectors**

While in general, warming temperatures are likely to make northern regions more hospitable for ticks, and possibly less so closer to the equator, direct effects on tick survival of increasing temperatures and changes in rainfall patterns on tick survival in many regions of the northern hemisphere may be limited, because of the protection afforded by the typical woodland habitats and also by their ability to undergo developmental and behavioural diapause to avoid unfavourable conditions [18].

Of greater impact on tick population survival is the expected effect of warming temperatures on rates of development from one life stage to the next, and on host-seeking activity. Because the duration of development from one life stage to the next is mostly temperature-dependent (within the constraints of diapause), warmer temperatures will probably mean shorter tick life cycles, and shorter development times will probably be coupled with extended periods of the year when temperatures are suitable for tick activity [36]. Laboratory experiments by Gilbert et al. [22] also suggest that a greater proportion of *I. ricinus* in the questing phase will become active as temperatures increase, and the interaction of temperature with humidity, driving the saturation deficit, also directly impacts host-seeking activity [37]. The success of host seeking can therefore be influenced directly by temperature effects on the ticks, but is also determined by the abundance and activity of hosts, which will be affected by the temperature-dependent availability of forage.

#### **5. Projected Effects of Climate Change on Tick and** *Babesia* **spp. Distributions**

With the future temperatures projected by climate models, it is expected that the northern limit of the range of *I. scapularis* will expand northwards [38,39] and the leading edge of this expansion is now north of the Canadian border (Figure 2).

**Figure 2.** (**A**) Maps of values of the basic reproduction number (*R*<sup>0</sup> ) of *Ixodes scapularis* in North America, estimated from ANUSPLIN observed temperature (1971–2000: upper panel), and projected climate obtained from the climate model CRCM4.2.3 following the SRES A2 greenhouse gas emission scenario for 2011–2040 (middle panel) and 2041–2070 (bottom panel). The colour scale indicates *R*<sup>0</sup> values. Temperature conditions that result in an *R*<sup>0</sup> of >1 permit survival of *I. scapularis* populations. Reproduced from Ogden et al., 2014 [38]. (**B**) Risk maps for the occurrence of *Ixodes scapularis* in Canada in response to increasing temperatures associated with climate change. The methods used to generate these maps are described by Ogden et al., 2008 [40].

Several studies in Europe have predicted a northwards expansion of the geographic range of *I. ricinus* [41–43], (for example see Figure 3).

**Figure 3.** Climate change prediction of *Ixodes ricinus* distribution in Scandinavia based on the length of the vegetation growth period, IPCC 2000 high emission scenario. Modified with permission from Jaenson and Lindgren, 2011 [41].

The models of Porretta et al. [42] and Alkishe et al. [43] also suggest that the distribution of *I. ricinus* is likely to extend eastwards, into habitat currently occupied by *I. persulcatus.* Additionally, *I. ricinus* is predicted to occur at increasingly higher altitudes in mountainous regions [44]. For the main tick vectors of *Babesia* spp. from the northern hemisphere, range expansion driven by climate would only be possible where suitable habitats occur. However, these tick species are, for the most part, woodland habitat generalists and as long as woodland habitats occur, it is likely that the ticks will survive in at least some of them, providing the woodlands also support host densities that are high enough. Some studies have suggested that southern range limits of ticks may contract northwards as more southern regions become too hot for ticks, particularly due to high temperatures inhibiting host-seeking tick activity [25,45]. Increased climate variability and extreme weather events (extreme heat and rainfall) may have relatively limited positive or negative impact on the ticks (compared to dipteran vectors) because of their relatively long multi-year life cycles and the capacity of their woodland habitats to provide an environment that protects the ticks from extreme weather [46]. Impacts of climate change on geographic ranges of hosts such as the white-footed mouse, *Peromyscus leucopus*, will likely have impacts on the geographic ranges and level of entomological risk of *B. microti* in current endemic areas [47]. It is possible that efficient host-to-tick transmission only occurs for a short period after initial infection [48] and if so, locations where there is seasonally synchronous activity of nymphal ticks (that infect the mice) and larval ticks (that acquire infection from mice), may pose a high risk. Effects of climate change on tick development and activity may cause changes to tick seasonality, resulting in locations where synchronous seasonal immature tick activity produce *B. microti* hot spots [23]. In addition to effects on synchrony, increased temperatures may result in changes in the proportions of the tick population feeding at different times of the year. Such an effect was observed in 1976 and 1977 in Ireland when an unusually hot summer in 1976 caused early activity of summer larvae, resulting in a marked increase in the proportion of nymphs active in the late autumn that year and the following spring [49], providing an indication of possible future effects of global warming.

Ticks themselves have very limited capacity for dispersal, and for any change in geographic range to occur, ticks and tick-borne pathogens including *Babesia* spp. need to be dispersed by ticks. Evidence suggests that two processes may be at play [50]—local dispersal that is likely by terrestrial hosts and breeding birds [51], and long-range dispersal by ticks carried on passerines that carry ticks northward in spring [40]. Some of these ticks may be infected with *B. microti*, but it is likely that dispersal by migratory birds is inefficient for *B. microti,* because this pathogen is not transmitted vertically by ticks (transovarial transmission) [30], and birds have, as yet, not been confirmed as reservoirs. As any larvae feeding on migratory birds would not be infected before or during their dispersal by birds, the only infected ticks that birds might carry would be nymphs infected as larvae on small mammal reservoir hosts, as demonstrated by the study of Wilhelmsson et al. [52]. Adult female ticks arising from such nymphs are likely to feed on deer rather than *B. microti* reservoir hosts and would pose little zoonotic risk since they would probably be free of infection following their second moult [30].

#### **6. Observed Effects of Climate Changes**

#### *6.1. Climate Effects on Ticks*

Studies in Canada suggest that the northern extent of the range of *I. scapularis* is determined by the limit of temperature conditions that allow ticks to complete their life cycles; i.e., when it is probable that an engorged, mated female tick gives rise to at least one other engorged, mated female tick (using the definition of Anderson and May [53] for a macroparasite, when the basic reproduction number of the tick is ≥1).

Combinations of data from active field surveillance for ticks, passive tick surveillance (involving detection of ticks at medical and veterinary clinics and by the public), and by inference from surveillance for human cases of tick-borne disease, such as Lyme borreliosis, have detected northern expansion of the range of *I. scapularis* [54–56] (Figure 4).

**Figure 4.** Surveillance for *Ixodes scapularis* populations in central and eastern Canada conducted from 2009 to 2015. Regions where *I. scapularis* populations have been identified by field surveillance are shown as red hatched areas. In 2004 there were only four known *I. scapularis* populations in locations shown by the red arrows. Tick populations have been identified in surveillance programs for Lyme disease (blue circles show municipalities where human Lyme disease cases have been identified). Infections due to *Babesia microti* are not yet nationally notifiable (reproduced with permission from Gasmi et al., 2017 [57]).

Figure 4 shows that *I. scapularis* was only detected at four locations in 2004 (red arrows), but that between 2009 and 2015 ticks and Lyme borreliosis cases had emerged in many other places. Surveillance data have detected a spatio-temporal pattern of range expansion of *I. scapularis* that is consistent with a warmer climate being a key determinant of range spread, and that support the accuracy of model-derived temperature thresholds for *I. scapularis* population survival (reviewed in Ogden et al. [23]). Furthermore, expansion of the tick range has occurred during a period of warming that is now considered a climate anomaly associated with anthropogenic climate change.

Similar trends have been observed for *I. ricinus*, particularly in Scandinavia [6,58,59], but also in Russia [60]. Additionally, the predicted altitudinal changes [23] in *I. ricinus* distribution have already been reported. In 1979 ticks were found up to 700 m a.s.l. in mountainous regions of the Czech Republic, but in 2002 were collected at 1100 m [61,62], and more recently (2020) have been found higher still at 1700 m in the Italian Alps [63]. An earlier study in 1993 had shown that *I. ricinus* was unable to complete its life cycle at such altitudes in the Czech Republic [64].

In Norway, Hvidsten et al. conducted one of the few surveys based on direct observation of *I. ricinus* occurrence at the northern limits of its distribution, collecting specimens by drag-sampling, small mammal trapping, from domestic animals, mainly dogs, and by mailed submissions [59]. Attempts were made to differentiate locations where *I. ricinus* populations were established from those where a few adventitious ticks had been observed or no ticks were detected. The criteria for tick establishment in a local region were the presence of all three life cycle stages in two successive years [65]. Estimates of the vegetation growing season length (VGSL), defined as the number of days when the mean temperature exceeds 5 ◦C, suggest that established tick populations occurred where the VGSL exceeded 170 days (Figure 5).

**Figure 5.** Vegetation growing season length (VGSL) in days in 1961–1990 and 1991–2015 correlated with the presence of *Ixodes ricinus* in northern Norway. The VGSL threshold for tick establishment was estimated to be approximately 170 days. Modified with permission from Hvidsten et al., 2020 [59].

The period of 170 days VGSL is the same minimum value for tick establishment estimated for Scandinavia by Jaenson and Lindgren [41] and was the basis for their projections of tick distribution changes over the next few decades (Figure 3). When the average VGSL

values in the Hvidsten et al. study for the period 1961–1990 are compared with those for 1991–2015, an increase in VGSL is evident at all locations, clearly associating the expanding tick distribution with rising temperatures. The most likely mechanism for this temperature effect is the time required for each tick stage to complete development within a season, as demonstrated by Daniel [64] in his altitude study, but low temperatures will also limit questing activity, and Gilbert et al. [22] have shown that *I. ricinus* nymphs from higher latitudes can quest at lower temperatures than those from more southerly regions.

#### *6.2. Climate Effects on Reservoir Hosts*

In addition to direct effects on ticks, rising temperatures will also affect their hosts, which is particularly important when these hosts serve as reservoir hosts for tick-borne pathogens. In the case of *B. divergens*, the pathogen's distribution is closely associated with that of cattle. Infections appear to result from local transmission by established tick populations [66], and since tick populations are expanding northwards, it is not surprising that there is some evidence, though indirect, for bovine babesiosis in more northern locations than in previous decades [67]. Since *B. divergens* is transmitted transovarially, infected larval or nymphal ticks could be deposited by birds far to the north of established tick populations. However, there is very little evidence for infections transmitted to cattle by such adventitious ticks, with only one suspected case in the far north of Norway occurring in the last 20 years [66]. A more definite climate effect on bovine babesiosis, though on a local scale, occurred recently in the south of England in February 2019, when temperatures exceeded the average for the time of year by more than 10 ◦C, causing very early tick activity and an outbreak of bovine babesiosis involving 20 cattle [68]. *Babesia venatorum* is also transmitted transovarially and is associated with roe deer, so presumably the distribution of this pathogen has been affected by the northward range expansion of its host [58]. It can also be distributed by birds carrying infected ticks, which led to speculation that its detection in sheep in Scotland may have resulted from the deposition of ticks by migratory birds from Norway [69]. At present there are insufficient data on the distribution of *B. venatorum* to associate it with any climate change effect.

Genotypes of *B. microti* have been detected in many vertebrate species but those that cause human babesiosis in north-eastern North America appear to be limited to the white-footed mouse, *Peromyscus leucopus*, short tailed shrews, *Blarina* spp., and chipmunks, *Tamia striatus*. Since, in the absence of transovarial transmission, neither birds nor deer can play a significant part in the introduction of the parasite to new reservoir host populations, and it appears that migration of infected small mammals is the main means by which the pathogen can emerge in new areas. *B. microti* infections therefore lag well behind the spread of other *I. scapularis*-borne diseases such as Lyme borreliosis and human granulocytic anaplasmosis [70], both of which can infect birds, though *B. microti* infections are spreading within the northeast and upper midwest endemic regions [50,71]. While the tick vector has spread north into Canada, human babesiosis remains exceedingly rare and to date only three cases of autochthonous infection have been recorded there [72].

There is little information on the effects of climate change on the small mammal reservoir hosts of *B. microti*, but intuitively one might expect milder winters to result in their improved survival, driving expansion of their populations. In *P. leucopus*, one of the main reservoir hosts, tick transmission is relatively inefficient, but appears to be facilitated by the agent of Lyme disease, *B. burgdorferi* sensu stricto [73], which is now prevalent in southern Canada and for which *P. leucopus* is also an important reservoir host. Furthermore, the persistence of *B. microti* in its rodent hosts, *Microtus* spp. and *P. leucopus*, is enhanced by vertical transmission [74,75], so range expansion of these rodents is likely to be fundamental to the spread of *B. microti.* Roy-Dufresne et al. [76] used an ecological niche factor analysis to study the potential effect of global warming on the distribution of *P. leucopus* and concluded that by 2050 the range of this rodent species could have expanded northwards by 3◦ latitude. Considering that the upper midwest *B. microti* endemic area is only just across the US border, it seems likely that significant numbers of

cases will eventually occur in Canada. Indeed, the three recorded autochthonous Canadian *B. microti* infections were apparently all acquired in southern Manitoba not very far from the endemic region in Minnesota [72], although unfortunately it is not known whether the same genotypes responsible for *B. microti* human babesiosis in the US were involved in these Canadian cases. An alternative explanation for the appearance of human babesiosis in new areas, is that zoonotic genotypes of *B. microti* occur in the absence of *I. scapularis*, being transmitted by tick species that generally do not bite humans, for example *Ixodes angustus.* While such cryptic cycles exist [77], there is little evidence so far that they have played a part in the spread of zoonotic babesiosis caused by *B. microti*, although they might have had a role in the establishment of the two separate foci in the northeast and upper midwest of the US, in which the *B. microti* genotypes show distinct differences from each other [78]. *Ixodes scapularis* (or *I. dammini*) is thought to have spread from coastal refugia in the 1950s as a result of reforestation and the growing deer population [79], and it is possible that in the upper midwest *B. microti* genotypes maintained in cryptic cycles were then able to infect this newly arrived bridge vector and thus establish a new focus of human babesiosis.

#### **7. Conclusions**

While observations suggest that tick populations have been responding to increasing temperatures with a northwards expansion for some years, it is not possible at present to be certain that the occurrence of human babesiosis has been affected by climate change. This is partly because of lack of data, particularly in Europe, where human babesiosis is much rarer than in North America, but also because the distributions of the pathogens involved depend on infected reservoir hosts, in addition to ticks, and the factors affecting the movements of these animals (small mammals, deer and domestic cattle) are influenced by other factors in addition to climate, notably landscape changes resulting from anthropocentric activity. Nevertheless, observations and models suggest that it is only a matter of time before human babesiosis cases occur more frequently, out of season and further north than at present as a result of climate change.

**Author Contributions:** Conceptualization, J.S.G.; writing—original draft preparation, J.S.G. and N.H.O.; writing—review and editing, J.S.G. and N.H.O. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

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

#### **References**


## *Review* **Human Babesiosis in Europe**

#### **Anke Hildebrandt 1,2 , Annetta Zintl <sup>3</sup> , Estrella Montero <sup>4</sup> , Klaus-Peter Hunfeld 5,6,7 and Jeremy Gray 8,\***


**Abstract:** Babesiosis is attracting increasing attention as a worldwide emerging zoonosis. The first case of human babesiosis in Europe was described in the late 1950s and since then more than 60 cases have been reported in Europe. While the disease is relatively rare in Europe, it is significant because the majority of cases present as life-threatening fulminant infections, mainly in immunocompromised patients. Although appearing clinically similar to human babesiosis elsewhere, particularly in the USA, most European forms of the disease are distinct entities, especially concerning epidemiology, human susceptibility to infection and clinical management. This paper describes the history of the disease and reviews all published cases that have occurred in Europe with regard to the identity and genetic characteristics of the etiological agents, pathogenesis, aspects of epidemiology including the eco-epidemiology of the vectors, the clinical courses of infection, diagnostic tools and clinical management and treatment.

**Keywords:** European babesiosis; *Babesia divergens*; *Babesia venatorum*; *Babesia microti*; *Ixodes ricinus*; parasite identity; epidemiology; clinical cases; diagnosis; treatment

## **1. History**

The first reported case of human babesiosis in Europe, and indeed in the world, occurred in 1956 in the former Yugoslavia, now Croatia, in a 33-year-old tailor and parttime farmer who had been splenectomized following a traffic accident 11 years earlier [1]. He presented with fever and severe hemoglobinuria eight days after first feeling unwell and died two days later. The parasites detected in blood smears were identified as *Babesia bovis.* However, *B. bovis* is not known to be zoonotic, and the photomicrographs in the published case report show divergent piroplasms that are characteristic of *Babesia divergens*, as well as a cattle parasite and first described by M'Fadyean and Stockman in 1911 [2]. The second recorded case, another *B. divergens* infection, which also ended fatally, occurred in 1967 in a splenectomized man who had apparently contracted the infection on holiday in the west of Ireland [3]. Further cases then followed in the 1970s in the UK and France, and to date, cases have been recorded in at least 19 European countries, almost always fulminant in splenectomized patients and attributed to *B. divergens*.

**Citation:** Hildebrandt, A.; Zintl, A.; Montero, E.; Hunfeld, K.-P.; Gray, J. Human Babesiosis in Europe. *Pathogens* **2021**, *10*, 1165. https:// doi.org/10.3390/pathogens10091165

Academic Editor: Cheryl Ann Lobo

Received: 1 August 2021 Accepted: 3 September 2021 Published: 9 September 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

A second zoonotic species emerged in 2003 in Italy and Austria [4], initially designated EU1, but now named *Babesia venatorum*. To date, infections with *B. venatorum* have been reported from Germany [5], Austria [6] and Sweden [7], all in splenectomized patients who survived, which possibly indicates a milder course of infection than *B. divergens*, though treatment has improved markedly since the first appearance of zoonotic babesiosis.

The most recent addition to the list of autochthonous zoonotic European *Babesia* spp. is *Babesia microti*, the first confirmed case of which occurred in Germany [8], and caused moderate illness in a spleen-intact but immunocompromised patient. A few mild or asymptomatic other cases have since been recorded, but it is very clear that the strains of *B. microti* present in Europe, where it is common in rodents and ticks, are not as infectious or pathogenic to humans as those in the USA, where *B. microti* infections give rise to approximately 2000 zoonotic babesiosis cases annually [9].

#### **2. Parasite Identity**

Two of the three *Babesia* species that infect humans in Europe, *B. divergens* and *B. venatorum,* belong to the *Babesia* sensu stricto (s.s.) group and are closely related, (Clade X; [10]), while the third species, *B. microti*, is phylogenetically distinct, belonging to *Babesia* sensu lato (s.l.) (Clade I; [10]). The three parasites are distinguishable morphologically in Giemsa-stained blood smears, but only by experienced diagnostic microscopists because they share important features. For example, divergent paired pyriforms are characteristic of both *B. divergens* and *B. venatorum*, while pyriform tetrads occur in both *B. divergens* and *B. microti.* The much more frequently observed single round trophozoite ('ring' stage) occurs in infections of all three species (Figure 1).

**Figure 1.** *Babesia divergens* in a Giemsa-stained thin blood smear. Round (**A**), paired pyriform (**B**), tetrad (Maltese cross) (**C**) and multiple parasite (**D**) forms are indicated. Similar round and paired pyriform forms have been observed in infections of *B. venatorum,* and round and tetrad forms occur in *B. microti* infections. Multiple parasite-infected erythrocytes are often seen in high parasitemias. © Estrella Montero, Luis Miguel Gonzalez.

*Babesia microti* can be distinguished serologically from *B. divergens* and *B. venatorum*, but the latter two are antigenically similar [4]. Serology is further limited by the time required for an antibody response to develop, which may be several weeks in immunocompromised patients [5]. DNA sequence discrimination is not only relatively swift, but can also be used to identify *Babesia* species, and it is now the method of choice for determining parasite identity. The 18S rRNA gene is by far the most commonly used locus for *Babesia* identification. There are several well-established and sensitive nested PCR protocols targeting this gene, as it is easy to amplify and there is much sequence information in the GenBank database.

It has long been accepted that cattle are the main, if not only, reservoir hosts for human *B. divergens* infections in Europe and sequencing of the 18S rRNA gene strongly supports this. Additionally, 5 of the 10 human *B. divergens* isolates for which sequence information is available are 100% identical with bovine isolate GenBank: U16370 (Figure 2), widely used as a reference sequence for *B. divergens* [11]. Another three isolates showed more than 99.9% homology with this bovine isolate reference, but a further two human isolates probably did not have a bovine origin, showing homologies of only 99.7% (GenBank: AF435415) and 99.2% (GenBank: AJ439713). The first of these cases occurred in a 34-yearold asplenic male Canary Island resident with typical clinical signs of acute babesiosis [12]. The patient reported having removed several unidentified ticks two weeks prior to being admitted to hospital. Since *I. ricinus* is apparently absent in the Canary Islands and the patient had no history of travel, the authors suggested the protozoan may have been transmitted by *Ixodes ventalloi*, an endophilic tick species that is common in the islands and primarily infects lagomorphs, carnivores and rodents [13]. However, this tick has never been confirmed as a vector for *B. divergens* or any other zoonotic *Babesia* species. The second case was reported in a 66-year-old acutely ill splenectomized patient in Portugal [14]. As the patient had travelled to Florida, the USA and the UK six weeks prior to his illness, it was not possible to ascertain where he had been infected.

There are several reports of the detection of *B. divergens* DNA in red deer (*Cervus elaphus*), roe deer (*Capreolus capreolus*) and reindeer (*Rangifer tarandus*) [15], and it has been suggested that these host species, especially red and roe deer, may serve as a source for infection for both humans and cattle. However, none of the deer isolates show 18S rRNA homologies of more than 99.9% with the bovine reference strain, and so far there is no evidence that deer '*Babesia divergens*' have ever caused either human or bovine babesiosis.

It is important to point out, that although useful for diagnostics, the 18S rRNA gene probably cannot unequivocally distinguish between *Babesia* species or strains. The reason for this is its highly conserved nature across the genus, on the one hand, combined with considerable intraspecific sequence diversity, on the other hand, particularly for *B. divergens* and *B. microti*. The cytochrome c oxidase subunit I (COI) gene locus has greater genetic diversity than the 18S rRNA gene and is therefore a more useful tool to distinguish between different species but, unfortunately, it has a lower amplification efficiency [16]. However, when examining the role of potential reservoir species, attempts should be made where possible to sequence sizeable fragments of both the COI and 18S rRNA genes [17]. Other loci such as the beta-tubulin, heat shock protein and merozoite surface protein genes have been used to distinguish *Babesia* isolates; however, until more information for these loci is available in the database, they are unlikely to be used more widely.

The clinical presentation of *B. venatorum* infections can closely resemble that of *B. divergens* [4], but this species is clearly distinct, showing only 98.2% homology with the bovine *B. divergens* reference sequence, U16370. Of the six infections reported in Europe so far, four 18S rRNA sequences are available, including the original two with identical sequences GenBank AY046575 [4], one showing 99.7% [5] and one showing 99.6% (asymptomatic–GenBank: KP072001) homology with AY046575. Almost all 18S rRNA sequences of *B. venatorum* from roe deer (*C. capreolus*) are identical to the first human isolates, with the few exceptions differing by one or two nucleotides, and there can be little doubt that roe deer is the main reservoir host of *B. venatorum* in Europe. It is also interesting to note that *B. venatorum* is not restricted to Europe and approximately 50 cases have been diagnosed in China [18], with the available 18S rRNA sequences being identical to the original European isolates or differing by only one or two nucleotides. The suspected but unconfirmed vector and reservoir hosts are *Ixodes persulcatus* and sika deer, or *Cervus nippon*, respectively [19].

**Figure 2.** The relative length, positions and heterologies of 18S rRNA sequences of *Babesia* species isolates from human cases compared to the reference sequence U16370. Numbers refer to positions in the reference sequence. Identity scores are according to Clustal Omega. '-' indicates that the base was missing.

*Babesia microti* is considered to be a species complex, mainly infecting small mammals. Goethert and Telford [20] assigned the parasites in the group to three clades based on analysis of the 18S rRNA and beta tubulin genes, with most of the zoonotic genotypes within Clade 1, which also includes the 'U.S. genotype' (e.g., GenBank: AY693840), responsible for the vast majority of human babesiosis cases. Human *B. microti* infections have expanded across the northeast of the USA over the last few decades [21], and several cases in Europe have been associated with travel from that country. In contrast, very few autochthonous cases have occurred in Europe, the first authenticated one was in Germany in an immunocompromised patient and caused by a strain (Jena–GenBank: EF413181) closely related to the USA genotype [8]. Welc-Faleciak et al. reported the detection of DNA of the same genotype in two other individuals, both asymptomatic, who were participating in a survey of forest workers in Poland [22]. Another *B. microti* strain, the 'Munich' type (GenBank: AB071177) is widely distributed in Europe and was originally presumed to be non-zoonotic [23]. However, DNA of this strain has reportedly been detected in seven patients in Europe, six of whom presented with various symptoms following a tick-bite in Poland (GenBank: KT429729; [24]), and one who presented with non-specific symptoms in Spain (GenBank: KT271759; [25]). A 157 nucleotide DNA fragment of the Munich strain was detected in an eighth patient, originally thought to be suffering from a prolonged bout of malaria while living in Equatorial Guinea. However, the patient made several visits to Spain during that period [26], and it is difficult to determine whether this *B. microti* infection, successfully treated with antibabesials, was contracted in Spain or Africa. All patients infected with the Munich strain were immunocompetent and only this latter patient had detectable parasites (at a very low level) in thin blood smears. These isolated reports indicate that two European genotypes of *B. microti* can infect humans, but that they are considerably less pathogenic than those in the USA. All cases that were imported into Europe appear to have an American origin (mainly North America), and although only three 18S rRNA sequences are available at present (showing 100% identity to the original American isolate GenBank: AY693840), it is probable that they were all caused by parasites closely related to this widespread U.S. genotype.

#### **3. Pathogenesis**

Most what is known to date on babesiosis pathobiology has resulted from in vitro experiments and animal studies (mainly in mice and cattle) on *B. microti*, *B. bovis* and *B. divergens* [27–31]. In many human infections, no isolates have been obtained for further investigations, and little information is available on the pathogenesis of *B. venatorum*. Babesia parasites occur within erythrocytes and as extracellular forms in the blood. They multiply within the erythrocytes by a form of budding to produce two or occasionally four daughter cells (merozoites). In fulminant human infections and in highly infected in vitro cultures, multiple parasites may occur within individual erythrocytes (Figure 1). The release of merozoites and eventual erythrocyte lysis is associated with fever and other clinical symptoms including hemolytic anemia, jaundice, hemoglobinuria, obstruction of renal arterioles and renal failure [32]. In vitro observations suggest that erythrocytes are not necessarily completely destroyed when the parasites leave them, but they are damaged. Their optical density decreases, they are reduced in size and are probably removed by the spleen soon afterwards [27–31]. While intermittent episodes of fever have been reported in cases of human babesiosis [32], they typically do not have the same regularity as febrile episodes in malaria, probably because of the asynchronous nature of babesia multiplication and egress from the erythrocytes [33]. In addition to erythrocyte lysis and metabolic alterations, excessive proinflammatory cytokine production contributes to clinical complications [34,35], potentially resulting in vascular leakage, adult respiratory distress syndrome, hypotension and shock [35,36].

Both innate and adaptive immune mechanisms limit the severity of babesial infection [34,37,38]. The spleen plays a central role in host defense by clearing infected erythrocytes from the bloodstream and mounting the protective immune response. The

heavily vascularized organ consists of red-pulp and white-pulp zones surrounded by a trabecula and an outer capsule. The marginal zone contains macrophages and neutrophils that recognize and ingest babesia-infected erythrocytes and circulating free parasites as the blood travels through the spleen (in humans, erythrocytes pass through the spleen approximately every 20 min [39]). The red-pulp infected erythrocytes are captured in sieve-like slits in the sinuses as they return to the main circulation and are ingested by macrophages [40,41]. The white pulp of the spleen contains T-cells that produce cytokines, for example gamma interferon (IFNγ), which activate macrophages to phagocytose and destroy parasites, as well as B-cells to secrete babesia-specific antibodies [41]. Antibodies neutralize pathogens, thereby preventing them from entering erythrocytes, and also enhance phagocytosis by macrophages and neutrophils through opsonization and eradicate pathogens through antibody-dependent cytotoxicity by natural killer cells and through the activation of complements [41]. The importance of cellular immunity in controlling parasitemia is demonstrated by the fact that both laboratory mice and humans with depressed cellular immunity have difficulties in controlling infections [5,28,31]. Similarly, the depletion of host macrophages and natural killer cells in mice increases susceptibility to infection [30], while an impaired antibody response due to hematological malignancies and/or rituximab therapy can also lead to difficulties in clearing infection in humans, despite adequate antibabesial therapy [5,42].

In general, factors responsible for severe infections following splenectomy include the delayed and impaired production of immunoglobulin and lack of splenic macrophages, resulting in a reduction in the numbers of infected erythrocytes removed [43]. Consequently, asplenia or hyposplenism often results in fulminant illness and death [1,44–47].

Babesia parasites possess a number of evasive measures to avoid immune attack, which can lead to persistent infections, even in the presence of an intact immune system [41]. Persistent infections (often asymptomatic) are particularly evident in *B. microti* infections of humans, but less so in *B. divergens* and *B. venatorum* infections, which are usually acute, although infection of their natural hosts (cattle and roe deer, respectively) tend to be persistent. The mechanisms for immunoevasion are unclear, although antigenic variation probably occurs to some extent. Capillary sequestration of infected erythrocytes, thus avoiding circulation through the spleen, has only been reported for certain non-zoonotic species (*B. bovis* and *B. canis*) [48].

#### **4. Vector Biology**

All zoonotic *Babesia* spp. in Europe are transmitted by the castor bean tick, *Ixodes ricinus.* This three-host tick species spends most of its life (>98%) free living, either host seeking or developing to the next stage. It requires a high humidity at the base of the vegetation (RH >80%), and ideal conditions are to be found in temperate deciduous woodlands with patches of dense vegetation and little air movement. Additionally, *I. ricinus* may be present in appreciable numbers in regions of high rainfall on agricultural land utilized by livestock, such as rough hill land or undergrazed pastures [49]. This tick species occurs in Northern, Western, Central and Eastern regions of Europe, but is sparse in Southern Europe because of its susceptibility to desiccation. In most regions of its distribution, host-seeking activity commences in spring and early summer, with ticks being found on vegetation and animals from late March and peaking in numbers from April to July. In some areas a second, less intense, phase of questing activity occurs in the autumn, and as a result of global warming, tick activity now occurs more frequently in winter [50]. All active stages of larva, nymph and adult ticks ambush their hosts from the vegetation and, with the exception of the male, which generally does not feed, they attach to the skin with specialized mouthparts for several days, the duration depending on the tick life cycle stage.

In infected unfed ticks, babesia parasites occur in the salivary glands, but they are not infective until they have undergone development, which is initiated when the tick starts to feed, and takes about two days to complete. *Ixodes ricinus* was first shown to be the vector of *B. divergens* in transmission experiments using splenectomized calves [51]. It appears

that infections are chiefly acquired by adult females while feeding on an infected host and they then pass the infection transovarially to the next generation of ticks, all stages of which (except perhaps for males) are capable of transmitting the infection [52]. While infection acquisition by immature stages has been suggested, this arose out of laboratory studies involving gerbils (*Meriones unguiculatus*) as hosts, and no direct evidence for the implied transstadial transmission that might follow exists [53]. *Ixodes ricinus* was also shown to be the likely vector of *B. venatorum* by Bonnet et al. [54], who demonstrated probable transovarial transmission from adult ticks feeding on roe deer to the next generation larvae. *Ixodes ricinus* as a vector of *B. venatorum* was validated in a subsequent in vitro study, in which both nymphs and females were shown to acquire infections and to transmit them transstadially and transovarially [55]. One of the consequences of transovarial transmission and transstadial persistence in *B. divergens* and *B. venatorum* is that theoretically infected ticks could occur in regions where infected reservoir hosts are not present, particularly as a result of the deposition of infected larvae by birds. Transstadial transmission of the third species, *B. microti*, involving acquisition by *I. ricinus* larvae from rodent hosts, followed by infection of hosts by nymphs, was reported by Walter and Weber in 1981 [56] and confirmed by Gray et al. in 2002 [57]. Additionally, the latter study showed that transovarial transmission of *B. microti* does not occur, that the parasite does not persist in the tick beyond one moult and that *I. ricinus* can transmit a zoonotic American strain, suggesting that it might be the vector of more than one European strain of the parasite.

Using PCR-based techniques, *Babesia* spp. are detectable in unfed free-living *I. ricinus* ticks. *Babesia divergens* occurs at a very low rate in ticks and is often undetectable even when the ticks have been collected from pastures where bovine babesiosis has occurred recently [58,59]. *Babesia venatorum* occurs at a slightly higher frequency [60], and as a parasite of roe deer, almost is always in ticks from woodlands. Tick infection rates of *B. microti*, also associated with rodents and woodlands, tend to be much higher, sometimes exceeding 10% [61].

Human *B. microti* babesiosis cases are exceedingly rare in Europe, despite the fact that this parasite occurs commonly in rodents and can be readily detected in unfed *I. ricinus* ticks [60], which are proven vectors of at least some Europe strains [57]. However, it should be noted that another tick species, *Ixodes trianguliceps*, is the dominant *B. microti* vector in many regions [62], and this tick species rarely bites humans. Furthermore, the parasites it transmits may not be infective for *I. ricinus*. These factors probably contribute to the low disease rate, but nevertheless, serosurveys indicate considerable exposure of the human population to infection [63].

#### **5. Epidemiology**

#### *5.1. Autochthonous Babesiosis Cases*

Human babesiosis is very rare in Europe, although the exact number of European cases is difficult to establish. Gorenflot et al. reported 22 cases of human babesiosis caused by *B. divergens* up to 1998 [64]. They occurred in France (10), the British Isles (6), Russia (1), Spain (2), Sweden (1), Switzerland (1) and present-day Croatia (1). Some cases were not published but communicated personally, and not all of them were confirmed using molecular methods [1,46,47,64–68]. From 1998 until the present, at least 13 additional cases were published in France [69–72], Portugal [14], Norway [73], Spain [74–76], Turkey [77], Finland [44], Ireland [78] and the UK [79] (Table 1). Overall, this amounts to more than 50 cases, with 35 attributed to *B. divergens*, 5 to *B. venatorum* and 11 to *B. microti* (excluding imported cases) (Tables 1–3).



**Table 1.** *Cont.*

tuberculosis, TBD—bacterial tick-borne disease, ARF—acute renal failure, ARDS—acute respiratory distress syndrome, HAP—hospital-acquired pneumonia, HLH—hemophagocytic lymphohistiocytosis. \* Classification of disease severity followed criteria suggested by Vannier and Krause (2009) [80]. \*\* infection probably acquired in Wales. AZM—azithromycin, ATQ—atovaquone, AP—atovaquone/proguanil, QN—quinine, CLI—clindamycin, VI—vibramycin, CTX—ceftriaxone, COX—cefotaxime, AMC—amikacin, OFX—olfloxacin, CHQ—chloroquine, PNT—pentamidine, CTM—cotrimoxazole, CH—chinin, MEF—mefloquine, ET—exchange transfusio


immunocompromizing treatment MI, some weeks/10 days 4.5% QN + CLI, AZM [8]

2015 Poland 2 patients >45 (NI) asymptomatic NI NI ND no treatment [22]

**Table 3.** *Cont.*


HLH—hemophagocytic lymphohistiocytosis, LGLL—low-grade lymphoplasmacytic lymphoma \* Classification of disease severity followed criteria suggested by Vannier and Krause (2009) [80]. T—tetracycline, AZM—azithromycin, ATQ—atovaquone, AP—atovaquone/proguanil, QN—quinine, CLI—clindamycin, CHQ—chloroquine, ET—exchange transfusion.

Despite the rarity of the disease in Europe, several serosurveys suggest that infections may be surprisingly frequent. For example, Hunfeld et al. reported positive values of 5.4% and 3.6% for *B. divergens* and *B. microti*, respectively, in a sample of 467 sera collected from the general population in Germany [63], and in Slovenia, IgG titers in 215 samples ranged from 8.4% to 2.8% depending on the IFAT cut-off [95]. However, in contrast to the USA, where transfusion-transmitted infections occur quite frequently, to date, only a single case in Europe (*B. microti* [8]) appears to have resulted from a blood transfusion. At the present time, therefore, and despite the increasing evidence for mild and asymptomatic infections [22,71,72] and the relative frequency of blood transfusions in the population, this form of transmission appears to carry a low risk of babesiosis in Europe.

The two greatest risk factors for zoonotic European babesiosis are exposure to *I. ricinus* ticks (though patients may not be aware of a tick bite) and splenectomy (Tables 1 and 2). Even in the few spleen-intact cases there was usually evidence of splenic dysfunction or other immune incompetence, as discussed below (Section 6.1). In contrast, the few European *B. microti* cases have all been spleen-intact cases. This is also a common feature in the many cases of infection with this parasite in the USA [96]. Considering the numbers of splenectomized individuals in the European population (several hundred thousand in France alone in 1983 [64]), and the abundance of *I. ricinus* throughout Europe, the frequency of babesiosis is surprisingly low, indicating perhaps that additional immunosuppressive conditions are contributing factors in disease occurrence (Table 1), but the low infection rates of ticks, even in habitats frequented by the relevant reservoir hosts, may also be a factor in the rarity of the disease.

Another obvious risk factor for *B. divergens* infection is association with cattle, as indicated by the genetic similarity between cattle isolates and those from human cases where the parasites were sequenced (Figure 2). Although *B. divergens* has been reported in red deer, the parasite genotypes differ from those isolated from cattle or humans (Figure 1, [15]). However, this topic requires further investigation, as discussed in the previous section. Since roe deer have been identified as reservoir hosts of *B. venatorum* [55], it is reasonable to suppose that exposure to ticks in woodland is a risk factor, as discussed in Section 4.

#### *5.2. Imported Babesiosis*

To date, 13 cases of human babesiosis have been documented as imported to European countries. All cases were attributed to *B. microti* and were diagnosed in Switzerland [83], the Czech Republic [84], Austria [85], France [87,92], Germany [86], Poland [82,88], Spain [90,91,94], Denmark [89] and the UK [93], having been acquired in the Americas (Table 3).

#### *5.3. Ambiguous Babesiosis Cases*

A small number of human babesiosis cases documented from Europe do not fit into any of the categories described above. These include three symptomatic cases with unidentified *Babesia* species reported in France [64,71] and Spain [64]. Additionally, one case of coinfection with *Babesia* spp. and *Borrelia* spp. was reported in Poland. Published sequences showed 98.99% homology to both *B. divergens* and *B. venatorum*, so that exact speciation was not possible [97]. In four more cases of babesia infection, which were detected in a retrospective study in immunocompetent patients in France, species identification was not possible [72]. Finally, one *B. microti* infection, diagnosed in Spain in a 43-year-old woman with an intact spleen, was associated with moderate and prolonged disease, originally diagnosed as malaria. Over an 8-month period, she received six consecutive diagnoses of malaria with different treatment regimens that led to no clear improvement. Because all antimalarial therapies failed, the patient's case was re-evaluated, diagnosed and eventually treated appropriately. It could not be established whether the patient acquired the infection in Europe (autochthonous) or in Equatorial Guinea (imported) [25].

#### *5.4. Reports of Possible Cases with Diagnostic Deficiencies/Lack of Clarity*

Of the 22 human cases of *B. divergens* infections that were documented before 1998 and described by Gorenflot et al. [64], and not all fulfill present day diagnostic standards, as some were only diagnosed microscopically without PCR confirmation, sequence analysis or serological testing [1,46,47,65–68,98,99]. Two cases reported as *B. bovis* infections were diagnosed on the basis of their appearance under the microscope only, rendering their identity questionable [1,100]. Even after 1998, at least 26 more cases were published as human babesiosis, despite diagnostic deficiencies or with lack of clarity, including 2 case reports of *B. microti* from Russia [101], 1 case from Spain [102] and 10 cases of unknown *Babesia* spp. from Montenegro [103], which were only diagnosed microscopically. Another case of *B. microti* infection in Switzerland presented doubtful microscopy, negative PCR and borderline serology [104]. A retrospective analysis of cases of babesiosis admitted to Spanish hospitals through data recorded in the minimum basic data set at discharge (MBDS) during the period 2004–2013 found 10 patients diagnosed with human babesiosis [105]. Only two of these were unequivocally identified as *B. divergens* and published [74,75]. Additionally, in a few cases, the co-infection of *Babesia* spp. with other tick-borne pathogens was reported, but unfortunately diagnoses in these cases were only performed on the basis of clinical presentation [105] or had deficiencies in the diagnosis of babesiosis [104,106]. For example, in a case of septic babesiosis reported from Spain [102], the patient presented a widespread exanthema with the presence of well-established annular lesions. Biopsy of one of the annular lesions showed changes compatible with a necrolytic migratory erythema. The patient had clinical symptoms of sepsis, but the diagnosis of human babesiosis was only based on apparent positive microscopy. There are no other reports of babesia infections causing erythema figuratum, and other differential causes have to be considered for this patient such as pancreatic neuroendocrine tumor-like glucagonoma, liver diseases and zinc deficiency [107–109]. Similarly, Strizova et al. reported a case of a 36-year-old man in the Czech Republic who experienced severe polytrauma requiring repetitive blood transfusions. Six months later he presented with possible Reiter's syndrome consisting of arthritis, conjunctivitis and urethritis. The diagnosis of human babesiosis caused by *B. microti* mimicking Reiter's syndrome was performed only based on apparent positive microscopy and the lymphocyte transformation test, which has not been evaluated for its sensitivity and specificity in the diagnosis of babesiosis [110]. Again, it is important to stress that the parasite is difficult to identify using microscopy alone, particularly if parasitemia is low, and confusion with platelets of staining artefacts is common (further discussed in Section 6).

#### **6. Clinical Course of Infections**

#### *6.1. Pre-Disposing Factors of Acute Disease*

General pre-disposing factors associated with a higher risk of symptomatic human babesia infection and more severe illness are splenectomy, impaired cellular and/or humoral immunity and advanced age [31,32,111]. The latter is explained by the decline in cellular immunity in patients over the age of 50 years [37].

In Europe, most severe cases were either splenectomized [1,4–7,14,45–47,70,73,74,77,86], or they had a rudimentary spleen and hyposplenism [44,78]. Immunosuppressive comorbidities associated with severe babesiosis are hematological malignancies such as Hodgkin's disease [4,5,45], B-cell lymphoma [4], acute myeloid leukemia [8], hairy cell leukemia [6], T-cell lymphoma [7] and HIV [74]. Patients with these malignant diagnoses and moderate-to-severe babesiosis were often on chemotherapeutic drugs with additional immunocompromising effects including prednisone, doxorubicin, cyclophosphamide, methotrexate, bleomycin and rituximab [4–8]. In human infections of *B. microti* in the United States, it has been reported that the severity of the disease increased with increasing parasitemia [112], with severe outcomes or complications of babesiosis associated with parasitemias of >4% [113] or >10% [114,115]. A few exceptions include reports of death in babesiosis patients with parasitemia <3% [116]. This latter observation

has also been made in critically ill patients in Europe [76], but in most European cases, parasitemias in patients with complications ranged from 10% up to 80% in *B. divergens* infection [14,44,46,70,75,77–79], 4% to 30% in *B. venatorum* infection [4,5] and 3% to 20% in *B. microti* infection [90–93].

One of the very rare but potentially fatal complications of babesiosis is hemophagocytic lymphohistiocytosis (HLH) [87,117–121]. In HLH, normal downregulation of activated macrophages and lymphocytes does not occur, resulting in excessive inflammation, hypercytokinemia, abnormal immune activation and tissue destruction. Dysregulation is due to the inability of natural killer cells and cytotoxic lymphocytes to eliminate activated macrophages. HLH is classified into a genetically determined primary form and a secondary form that occurs in older people with underlying conditions such as infections, malignancies and autoimmune disorders [122]. In Europe, HLH has been reported in at least four patients with babesiosis caused by *B. divergens* [74,123], *B. venatorum* [7] and *B. microti* [87]. Cofactors for severe disease in these patients were older age [87], and a newly diagnosed HIV infection in one patient [74].

In many European cases, detailed laboratory parameters are frequently not available, so that a retrospective analysis of potential clinical factors that may have rendered patients to be more susceptible is not feasible. Future reports on European cases should consider risk factors for severe disease that have been reported for *B. microti* infection including hemoglobin level <10 g/dL, parasitemia ≥10 days, elevated alkaline phosphatase >125 U/L, total white blood cell count >5 <sup>×</sup> <sup>10</sup>9/L and prior existing cardiac abnormalities [113,114].

#### *6.2. Babesia divergens*

This section describes the clinical course of babesiosis cases reported over the last 21 years (2000–2021) in asplenic, hyposplenic and normosplenic patients, who presented with mild-to-severe disease, dependent on age, immune status and co-morbidities (Table 1).

#### 6.2.1. Features of the Disease in Asplenic and Hyposplenic Patients

Since 2000, six patients who had been immunocompromised by splenectomy developed severe infections [14,69,70,73,74,77]. Two other patients had rudimentary spleens probably resulting in functional aspleny [44,78]. The period from onset of symptoms to the diagnosis of babesiosis ranged from one month prior to admission to four days post admission. Before obtaining a correct diagnosis of human babesiosis, patients were misdiagnosed with malaria, fever of unknown origin and *Mycobacterium* spp. infection secondary to HIV. Frequently presented symptoms were a fever up to 40 ◦C, headache, abdominal and back pain, fatigue, hemolysis with or without anemia and jaundice. Severely ill patients developed acute renal failure, hemophagocytic syndrome, atrial fibrillation, ARDS, hospital-acquired pneumonia, pulmonary aspergillosis, septic shock and multiorgan failure [14,44,69,70,73,74,77,78]. Parasitemias ranged from 1% [69] to 60% [70]. Only two of these eight patients (25.0%), a 66-year-old man [14] and a 53-year-old man [44], succumbed to the infection indicating a significant improvement in survival rates compared to cases reported before 1998 [64].

Interestingly, the two patients who were not splenectomized but were hyposplenic developed very severe disease. One case involved a 79-year-old man in Ireland with a 5-day history of fever, malaise, nausea, generalized pains and dark-colored urine. The patient remembered removing a tick from his arm two weeks prior to the onset of illness. He had been diagnosed with celiac disease several years before admission. A peripheral blood smear revealed babesiosis with 20% parasitemia and the presence of Howell–Jolly bodies. The patient received antibabesial treatment but developed several complications including acute renal failure, ARDS and hospital-acquired pneumonia. Altogether he was hospitalized for 61 days. An MRI scan later revealed an atrophic spleen [78]. Defective splenic function affects more than one-third of adult patients with celiac disease [124]. Eliminating gluten from the diet may improve splenic function [125], but this works

inconsistently and apparently not in those patients who have already developed splenic atrophy [125,126].

The second hyposplenic patient was a 53-year-old man from Finland who succumbed to the infection. He showed typical symptoms, but also had dark streaks on his arms and legs, probably caused by massive intravascular hemolysis, and an erythema migrans indicating possible co-infection with *Borrelia burgdorferi* s.l. On post-mortem examination, splenic atrophy was found, probably caused by alcohol consumption and/or by a previous history of alcohol-induced pancreatitis. Unfortunately, no mention was made of any investigation for other possible causes of this case of hyposplenism, such as celiac disease or other autoimmune diseases [44].

#### 6.2.2. Features of the Disease in Normosplenic Patients

*Babesia divergens* parasitemias in immunocompetent individuals are generally lower than in immunocompromised patients and are often difficult to detect [71]), but at least six cases of infection have been reported in normosplenic patients during the last 21 years [71,72,75,76,79]. The course of disease ranged from mild [71] to severe [75,79] and even lethal [76], and parasitemias ranged from 0.29% [71] to 20% [78,79]. Mild cases presented with fever, chills, headache, arthromyalgia, leukopenia and elevated liver enzymes [71,72]. More severely ill patients had fever, malaise, vomiting, abdominal pain, hemolytic anemia, jaundice, hemoglobinuria and acute renal failure [75,76]. One of these six patients (16.7%) died [76].

One of the cases involved a 46-year-old forest ranger in Spain who was hospitalized after 3 days of fever, severe abdominal pain, jaundice and black and red deposits in his urine. Laboratory parameters indicated hemolytic anemia. CD4+ T cell counts were normal and serologic tests and blood cultures for hepatitis and HIV, as well as *Bartonella*, *Brucella*, *Leishmania*, *Leptospira* and *Borrelia* spp. were negative. Initial parasitemia was 10%, diminished gradually and resolved 10 days after starting a 12-day course of antibabesial therapy of quinine and clindamycin. Interestingly, hemolytic anemia remained severe, as evidenced by low hemoglobin. The patient's illness unexpectedly relapsed on day 18 after treatment. Parasites were again detected in blood samples and he was put on a 7-week course of combined atovaquone/proguanil and azithromycin [75].

In another case, a 72-year-old immunocompetent patient in the UK developed a parasitemia up to 20%. Unfortunately, we have no information about the clinical course of the disease in this patient. Older age was the only known risk factor [79]. Old age was probably also a factor in a fatal *B. divergens* infection in an 87-year-old woman from Spain who was hospitalized after three months of low-grade fever, malaise, vomiting, decreased appetite, jaundice and hemoglobinuria. In her case, parasitemia was low (2.9%). Although she received effective antibabesial treatment that cleared the parasites by day 15 following admission, she developed acute renal failure, nose and mouth bleeding and extensive cutaneous hematomas as result of disseminated intravascular coagulation, which resulted in death [76]. In addition to her advanced age, the patient also had complex cardiovascular co-morbidities, which in *B. microti* infections have been identified as risk factors for severe disease [113].

The first indication that *B. divergens* may cause relatively mild infections was reported by Martinot et al. in 2011 in France [72]. They detected intraerythrocytic parasites and *B. divergens* DNA in a 37-year-old woman with an unremarkable medical history, who presented with fever, headache and arthromyalgia two weeks after a tick bite and who recovered without specific antibabesial medication. Infected erythrocytes were also observed in a 35-year-old man showing similar symptoms, who also recovered uneventfully, but it was not possible to speciate this parasite using PCR. More recently (2018), also in France, Paleau and others detected *Babesia* spp. infection in six patients with flu-like symptoms, using a combination of tests that included PCR [72]. *Babesia divergens* was definitively identified in two of the cases. Interestingly, one patient was additionally diagnosed with *K. pneumonia* septicemia and hepatic abscesses, perhaps indicating an unrelated

co-infection or a superinfection of acute or chronic human babesiosis. Another patient was diagnosed additionally with hemolytic anemia and acute pneumonia. Although pulmonary symptoms have been described in relation to human babesiosis, an unrelated co-infection could not be ruled out. Finally, babesiosis was diagnosed in a patient presenting with febrile eosinophilic panniculitis, which is an unusual cutaneous symptom in babesiosis. Unfortunately, there is no information on the patient's history, medication or co-morbidities [72].

#### *6.3. Babesia venatorum*

Altogether, five cases of *B. venatorum* have been described in Europe to date, in Austria [4,6], Italy [4], Germany [5] and Sweden [7] (Table 2). An additional unpublished case from Poland, listed in GenBank under accession number KP072001, is not discussed in this section. Interestingly, the five patients were over 50 years of age, splenectomized and diagnosed with hematological malignancies including Hodgkin's disease [4,5,7] and hairy cell leukemia in the fifth [6]. One of the Hodgkin's patients also had large B-cell lymphoma [4]. Four patients received immunosuppressive drugs including bleomycin [4], prednisolone + rituximab [5], methotrexate [6] and cyclosporine + prednisolone [5]. One patient developed mild [4], one patient mild-to-moderate [6] and three patients moderateto-severe [4,5,7] disease. Reported symptoms were recurrent episodes of fever, progressive weakness, shortness of breath, thrombocytopenia, jaundice, abdominal pain, hemolytic anemia with elevated serum lactate dehydrogenase, elevated indirect bilirubin values, low haptoglobin levels and acute renal failure with dark urine as result of hemoglobinuria [4–7]. In two patients, a positive direct Coombs test led to an initial misdiagnosis of autoimmune hemolytic anemia potentially due to ongoing Hodgkin's disease [5] or ongoing hairy cell leukemia [6]. Moreover, elevated C-reactive protein and procalcitonin levels suggested persistent infection in two patients [5,7]. Bone marrow examination of the Swedish patients showed a few phagocytosing macrophages and monocytosis leading to a tentative diagnosis of hemophagocytic lymphohistiocytosis with supporting laboratory evidence including elevated triglycerides, ferritin and soluble interleukin-2- receptor [7]. Parasitemias in the *B. venatorum* infections ranged between 1.3% [4] and 30% [4,6]. While all patients eventually seroconverted [4–7], the German case remained seronegative for specific antibodies for several months and suffered a relapse after the conclusion of the initial treatment. Moreover, retreatment with atovaquone and azithromyin for two months was unsuccessful in clearing the parasite, and low-level parasitemia persisted for several months despite maintenance therapy with atovaquone, possibly due to the previous combined application of rituximab and prednisolone, which have highly immunosuppressive effects. The Swedish patient also had fluctuating parasitemia for several months, although it was not clear whether this was a natural feature of the infection or due to injections with human immunoglobulin [7]. All five patients were cured [4–7]. A study in China on people who sought medical help after a tick bite detected 48 out of 2912 individuals with *B. venatorum* infections [127], suggesting that cases caused by this parasite generally take a milder course than those caused by *B. divergens*, requiring special awareness for detection and appropriate treatment.

#### *6.4. Babesia microti*

#### 6.4.1. Autochthonous *B. microti* Infections

*Babesia microti* infections in humans are rarely reported outside the United States. So far, only 11 autochthonous cases have been reported from Europe (Table 3). A marked characteristic of *B. microti* infections, in contrast to those caused by *B. divergens* and *B. venatorum*, is that the vast majority of cases have occurred in normosplenic patients. Moreover, asymptomatic infections appear to be common. However, clinical manifestations in asplenic patients are very similar to those caused by *B. divergens* and *B. venatorum*, often fulminating and resulting in death [32,42,113,114,128,129]. Patients who have recovered from acute babesiosis often maintain persistent asymptomatic parasitemia lasting for several

months. In immunocompromised individuals, *B. microti* infections may even persist through multiple courses of treatment [42,130]. Relapse of illness is also more common in immunocompromised than previously healthy adults, but even in this group it may occur as long as 27 months after the initial illness [131,132]. Since many infections are asymptomatic and/or persistent, transmission of *B. microti* through blood transfusion is a serious public health threat in the USA [32,133]. Transfusion-related transmission may arise at any time of the year and incubation periods can be much longer than in tick-transmitted infection [134,135].

The first reported European case of *B. microti* occurred in Belgium in an otherwise healthy man in his 40s in Belgium in 1981 [81]. He suffered from fever and weight loss of 8 kg within one month. His serum was reactive for *R. conori* and *B. microti* and *B. rodhaini*. The patient was cured, but it is not clear whether he was infected with *B. microti* or if antibodies showed cross-reactivity with *Rickettsia* spp. [81]. The first validated case occurred in Germany [8] and is the only one so far in which parasites were observed within erythrocytes. The other nine documented cases occurred within the last six years in Poland [22,24] and Spain [25] and were diagnosed by the detection of parasite DNA. All patients had an intact spleen, and in all but one patient immunocompetence could be assumed. The exception was the German case, who was immunocompromised because of treatment for myeloid leukemia. In this case, a moderate disease developed with fever, heavy chest pain, hypertension, tachycardia and pancytopenia. Microscopy showed an initial parasitemia of 4.5% [8]. However, it is difficult to determine whether pancytopenia resulted from the *B. microti* infection, the underlying disease of acute myeloid leukemia or a combination of both. It is notable that this patient showed acute onset of babesiosis with clinical symptoms of coronary heart disease, probably due to ongoing anemia. Acute disease manifestation was followed by subsequent seroconversion for *B. microti*-specific antibodies six weeks later and points to a newly acquired infection rather than an acute exacerbation of a pre-existing subclinical parasitemia. The specific antibody response disappeared four weeks after seroconversion, probably owing to the start of another cycle of chemotherapy with cytarabine and idarubicin. The source of infection in this case was apparently an infected blood transfusion from an asymptomatic blood donor [8], whereas in the other patients tick-bite transmission is probable. The other patients with *B. microti* infections included two individuals, who were randomly identified as part of a study of forestry workers, employed in the Podlaskie province of Eastern Poland. Both were >45-years-old adults and reported several tick bites while working in forests over the preceding two years [22]. Six patients in Poland and one patient in Spain had a mild disease with nonspecific clinical symptoms such as fever, muscle pain, joint pain, headache, vertigo, fatigue and general malaise [24,25]. The case in Spain is an example of low-grade chronic human babesiosis caused by *B. microti*, with intermittent symptoms for a period of at least four months. Such cases may go undiagnosed in immunocompetent patients [25]. Altogether, four patients seroconverted and all those with symptoms were cured.

#### 6.4.2. Imported *B. microti* Infections

Parasitemias of imported cases ranged up to 20% [90,91,93] (Table 3). Clinical symptoms were similar to those of autochthonous cases characterized by fever, fatigue, malaise, chills and headache, as well as signs of hemolytic anemia, thrombocytopenia, acute renal failure and multiorgan failure in severe cases [90,91,93]. Unusual symptoms were neck stiffness in a patient with additionally diagnosed neuroborreliosis [88], and lower back pain, continuous knee pain and erythematous skin changes without any detected co-infection [89]. Bone marrow aspiration of a patient with severe pancytopenia showed typical hemophagocytosis [87]. Although all 13 patients with imported *B. microti* infections were evidently in good health for travel [82–94], an 83-year-old man diagnosed with lowgrade lymphoplasmacytic lymphoma died of the infection [93]. *Babesia microti* infection should definitely be a differential diagnosis in Europe, especially for patients with a travel history to the Americas.

#### **7. Laboratory Diagnostics**

As human babesiosis can take a fulminant course of disease, especially in immunocompromised patients infected with *B. divergens*, rapid diagnosis is essential. A study of patients infected with *B. microti* reported that cases where diagnosis was delayed for 7 days or more were significantly associated with more severe disease [115]. In Europe, misdiagnoses (malaria, autoimmune hemolytic anemia with positive Coombs test) and lack of awareness of the existence of *Babesia* spp. as a causative infective agent have occasionally led to delayed diagnosis in the past, resulting in prolonged and potentially life-threatening disease [1,14,25,68,85] (Table 1, Table 2, Table 3). Indeed, in some cases, human babesiosis was only diagnosed post mortem [1,68]. We strongly recommend that diagnostic procedures for babesiosis should be initiated in patients that present with intermittent fever, fever of unknown origin or signs of hemolytic anemia. Patient records should include information on potential immunocompromising conditions, exposure to ticks, having received blood transfusions and travel to the USA or China within the last 6 months.

Clinical laboratory diagnosis of human babesiosis is challenging and it is uncertain whether automated hematology analyzers can reliably detect piroplasms. Where there are typical clinical symptoms, a positive Coombs test in combination with hemolytic anemia and elevated procalcitonine levels is highly suggestive of babesiosis and should prompt further diagnostic testing [5,136].

#### *7.1. Light Microscopy*

Ideally, direct pathogen detection is recommended for a definitive diagnosis. The gold standard is microscopic detection in a Giemsa or Romanowsky stained blood smears [111,137]. However, early in the course of infection or because of a low-level parasitemia, parasites may be difficult to find and smears from serial blood collection must be investigated [80,137,138]. Malaria is the most important differential diagnosis because the early stages of *Plasmodium* spp. intraerythrocytic ring forms lack the parasite pigment (hemozoin) that occurs in later stages, and thus resemble the round forms of *Babesia* spp. Hence, reliable *Babesia* spp. identification is not possible microscopically unless paired pyriforms or tetrads (Maltese crosses) are seen [111]. Piroplasms appearing in thin blood smears are ring- or pear-shaped forms with reddish chromatin and slightly bluish cytoplasm (Figure 1). Babesia merozoites arranged as tetrads usually occur in cases where there is a high parasitemia and are mainly observed in Clade 1 *Babesia* spp. (*B. microti*, *B. duncani*), but also in *B. divergens*. Parasitemias can range from <1% to 80% of infected erythrocytes and are mostly low in immunocompetent patients and at the onset of disease. Therefore, a thorough evaluation of ≥300 fields of vision and serial preparation of multiple smears is recommended [111,137]. It is important to stress that for species identification, microscopical detection of parasites in blood smears without additional molecular analysis of the pathogen is not sufficient.

#### *7.2. Molecular Diagnostics*

Nucleic acid testing is usually performed as a PCR targeting the 18S rRNA gene. This test is sensitive and specific in detecting *Babesia* spp. from clotted or EDTA blood. Sequencing of the 18S rRNA gene can be used for species identification, which has an epidemiological and therapeutic significance. The detection limit is approx. 1–3 parasites/µL of blood, and thus below that of microscopic methods [139]. There are various modifications of the test format and the molecular target structure including DNA/RNA hybridization (e.g., FISH), and real-time PCR methods [4,139], but there is currently no commercial test or sufficiently validated protocol available in Europe for diagnosis confirmation by a broadly accepted gold standard test [111].

#### *7.3. Culture*

*Babesia divergens*, *B. microti* and *B. duncani* can be cultivated in gerbils, mice and hamsters, respectively, while *B. venatorum* has not yet been adapted to a laboratory animal

species. Approximately 0.5–1 mL of EDTA or heparin anticoagulated whole blood are inoculated intraperitoneally, and the animal blood is monitored at least once a week for up to two months. Parasitemia is detectable after one week at the earliest but can be reliably detected after up to four weeks. There are many reasons why xenodiagnosis is impracticable in routine laboratories (e.g., labor-intensive and time-consuming process, availability, ethics and sensitivity). Likewise, the in vitro cultivation of piroplasms, which is possible in principle, requires sophisticated techniques, and is thus labor intensive and costly. Having in mind these practical drawbacks, culturing is reserved for specialized laboratories, although a broader approach to cultivating more isolates both from the veterinarian and the human medical fields is clearly desirable [111,140].

#### *7.4. Infection Serology*

The indirect immunofluorescence assay is the most commonly used serological test method. Cut-off titers for IgG antibodies from 1:32 to 1:160 were found to be sensitive (>88%) and specific (>90%) in multicenter studies with *B. microti* and *B. divergens* antigens [63,141]. However, cut-off titers should be adjusted to the local seroepidemiological situation and circulating *Babesia* species [63]. IgG titers of ≥1:1028 occur during the course of infection, which then decrease to titers of 1:64 within months to years. IgG assays do not reliably differentiate between acute, chronic or past infections [63,111,136,141]. On the other hand, IgM antibodies are detectable from approx. two weeks after the onset of symptoms onwards and indicate acute infection [141,142]. However, since false IgM-positive test results are common, particularly as part of untargeted testing in non-endemic areas, a two-step procedure is required in which only IgG-positive samples are further tested for the presence of IgM antibodies [63,140]. Assays that detect anti-*B. microti* antibodies do not detect antibodies against *B. duncani*, *B. divergens* or *B. venatorum* [143]. In contrast, crossreactivity between *B. divergens* and *B. venatorum* can be exploited diagnostically [5,137].

In addition to the general limitations of immunofluorescence assays (unknown test quality, investigator dependent variability, etc.), false-positive reactions have been described in sera from rheumatic patients and from patients with other, especially closely related infectious diseases such as malaria and toxoplasmosis [63,143]. Furthermore, the antibody response may not yet be present in the early phase as shown in acute European case reports or may be absent in immunocompromised individuals [5,136,140]. Therefore, it is not suitable for acute diagnosis but primarily for epidemiological purposes. Several publications describe other immunoassay formats (e.g., enzyme immunoassays, bead-based assays or immunoblots) that use a wide variety of antigens [140]. However, standardized serological test methods that have been validated by multicenter studies are currently not available in Europe due to low demand and lack of diagnostic evaluation.

Finally, it should be stated that, except for research and surveillance purposes, the practice of generally applying multiplex approaches for molecular diagnostics and/or serology in patients after a tick bite or in individuals with suspected Lyme borreliosis is not recommended, because from a statistical stand point, such diagnostic regimes will end up with many false-positive test results given the generally low incidence of tick-borne infections other than Lyme borreliosis in most European countries.

#### **8. Clinical Management**

Several drugs are available for the treatment of human babesiosis (Table 4), but their efficacy is variable, particularly against *B. microti*, which animal studies suggest is less susceptible to classic antibabesials than are *B. divergens* and *B. venatorum* [144]. However, available information on antibabesial susceptibility from case reports and clinical investigations suggests that there is no convincing scientific evidence for any clinically relevant differences in the susceptibilities of the pathogenic *Babesia* spp. to the therapeutic agents commonly used to treat human babesiosis [111,145]. Nevertheless, there is room for improvement in drug efficacy, particularly in relation to side effects, drug resistance and speed of response. In the case of most infections caused by *B. divergens* and *B. venatorum*, as well

as severe cases of *B. microti* infection, the speed of response to antibabesial administration is particularly important, and adjunct measures are often necessary. Most of the recent cases of human babesiosis caused by previously unknown *Babesia* spp. have responded to antibabesials used against known species [111,145]. However, until further data become available, treatment of infections caused by unknown *Babesia* spp. should include close monitoring of the course of parasitemia and long-term follow-up of such patients.

**Table 4.** Commonly and experimentally used drugs for the treatment of human babesiosis (modified from Hildebrandt et al., 2013 [111]).


a In immunocompromised patients, higher initial doses (600–1000 mg/day) may be required. <sup>b</sup> In immunocompromised patients, higher dose may be required. **<sup>c</sup>** maximum: 650 mg per dose. <sup>d</sup> maximum: 600 mg per dose, <sup>e</sup> maximum: 250 mg per dose. **<sup>f</sup>** maximum: 750 mg per dose. <sup>g</sup> In addition to standard drugs, alternative substances have been used successfully in some severe adult cases of babesiosis (see also Table 3) (111). <sup>h</sup> Imidocarb dipropionate is not licensed for use in humans. The dosing regimen for treatment of human babesiosis is derived from two successfully treated Irish cases with *B. divergens* infections (146).

#### *8.1. Babesia divergens*

Although sporadically observed in immunocompetent patients with viral-like illnesses, clinical cases of *B. divergens* have almost always been reported in asplenic or spleen-impaired individuals [71,78,111]. Many *B. divergens* infections in the past ended fatally with general organ failure occurring four to seven days after the initial presentation of hemoglobinuria. Outcome data in severely ill asplenic individuals show a mortality rate of 42% [27,70,71,137,146]. Consequently, the status of asplenic *B. divergens*-infected patients is regarded as a medical emergency, requiring immediate treatment to arrest hemolysis and prevent complications [111,137]. The combination of clindamycin and quinine for 7 to 10 days (Table 5) dramatically improves disease outcome [137,146–149], but in recent years, a more favorable disease course has been increasingly reported for *B. divergens*-infected patients, including those not treated with a full course of clindamycin and quinine because of quinine side effects [70,111,150]. These findings underscore the impact of improved adjunctive measures provided by modern intensive care medicine, including exchange transfusion [111,137]. This measure is usually reserved only for the most extremely ill *B. microti*-infected patients but has also been recommended for all severe *B. divergens* cases [27,111,137,145]. Alternative treatment options for *B. divergens* infections have included clindamycin monotherapy or imidocarb in conjunction with the above-mentioned adjunctive measures (Table 4) [69,102,111,137,151]. Imidocarb, one of the most effective antimicrobials for use in *Babesia*-infected animals, is highly active against this organism

in vitro [152]. It was used successfully to treat two Irish patients infected with *B. divergens* but is not licensed for use in humans [153]. Atovaquone proved more effective than imidocarb in an experimental *B. divergens* gerbil model and perhaps should be considered in combination with azithromycin for treatment of *B. divergens* infections and more generally for those caused by any *Babesia* s.s. species [152]. Atovaquone, together with either azithromycin or proguanil, has been used in three recent cases, following problems with toxicity or inadequate efficacy of other drug regimens [74–76], and it resulted in patient recovery in two of them [74,75].

**Table 5.** Commonly used drug combinations and treatment alternatives for human babesiosis with regard to parasite species and severity of the disease (adapted and modified from Hildebrandt et al., 2013 [111]).


<sup>a</sup> Usual duration of treatment is 7–10 days. Longer treatment (>6 weeks) may be necessary in immunocompromised or relapsed patients. In immunocompromised individuals, reduction of immunosuppressive therapy may be needed if possible for clearing the parasite. <sup>b</sup> Severe illness criteria according to White et al., 1998 [113]: parasitemia > 4%, alkaline phosphatase >125 U/L and white blood cell counts >5 <sup>×</sup> <sup>10</sup>9/L. Partial or complete exchange transfusion is recommended in case of high parasitemia (>10%), severe anemia (<10 g/dL) and pulmonary or hepatic failure. In severe disease cases i.v. treatment is suggested. Alternative treatments as derived from single case reports or case studies cited in the literature (Hildebrandt et al., 2013 [111]).

Although quinine, clindamycin, atovaquone and azithromycin, and some in combination, are proven antibabesials for the treatment of *B. divergens* infections in humans, there are concerns about rapid efficacy, drug resistance and recrudescent infections. However, cases do not occur frequently enough to justify research in drug discovery and development for human treatment alone. In recent years a significant number of drugs have been tested against this parasite in vitro for veterinary use, for example atranorin [154], cryptolepine [155], fusidic acid [156], hydroxyurea and eflornithine [155], myrrh oil [157] trans-chalcone and chalcone 4 hydrate [158], and the hope is that promising drugs will also prove useful for human infections.

#### *8.2. Babesia venatorum*

In general clindamycin with or without quinine and with or without subsequent combined atovaquone and azithromycin treatment have been used successfully in European cases of *B. venatorum* infection [5–7]. Problems with speed of response to therapy and parasite persistence occurred in one case [5].

In contrast to the more sporadic occurrence of *B. venatorum* cases in Europe, the disease is endemic in northwestern China with more than 48 reported cases [159–162], all of which were immunocompetent, in contrast to European patients. In these cases, 4 of the 48 Chinese patients received clindamycin alone and no deaths were reported [162]. Although the clinical course of *B. venatorum* generally seems to be milder than that of *B. divergens*, clinicians should be aware that immunocompromised patients might experience relapse and persistence of infection despite antimicrobial treatment. In such cases, it is important to monitor parasitemia by blood smear examination and PCR analysis and provide long-term clinical follow-up [5,111].

#### *8.3. Babesia microti*

Autochthonous *B. microti* infections in Europe are rare and most cases have been reported in travelers, mainly those returning from the USA. In such cases, treatment should follow American standards [145]. Animal studies showed that regimes of azithromycin in combination with quinine [163], azithromycin with atovaquone [164] and atovaquone with clindamycin [144] were all effective (Tables 4 and 5).

Randomized trials in humans infected with *B. microti* showed that atovaquone plus azithromycin therapy was as effective as the standard quinine/clindamycin combination and there were fewer side effects (15% versus 72%) [165]. In view of the low risk of side effects associated with atovaquone/azithromycin, it has been argued that all patients diagnosed with *B. microti* infection should be treated with this drug combination [111,137]. In severe cases, similar adjunctive measures to those used for *B. divergens* infections may be necessary [111] (Table 5).

Major obstacles to the development of new drugs against *B. microti* are, firstly, that a continuous in vitro culture system is lacking for this parasite despite much research on the topic, and secondly, that although continuous culture systems already exist for *Babesia* s.s. species such as *B. divergens* [152], antibabesials developed against these parasites appear to be relatively ineffective against *B. microti* [144]. However, the recent successful development of continuous in vitro culture systems for *Babesia duncani*, using human or hamster erythrocytes [166,167] promises progress in this area since *B. duncani* is more closely related to *B. microti* than to the *Babesia* s.s. species [10].

#### *8.4. Exchange Transfusion Management*

Exchange transfusion has been recommended for severe *B. microti* infection characterized by parasitemias of more than 10%, and/or severe anemia (hemoglobin <10 g/dL) and/or evidence of organ dysfunction (hepatic, pulmonary or renal compromise), as well as for all emergency cases involving *B. divergens* [111,137,145]. Such a procedure can contribute to the rapid reduction of parasitemia, correction of anemia and elimination of toxins and harmful metabolites, but it is complex and should take place under the supervision of specialised hematologists, taking into account the status and co-morbidities of the patient. Although erythrocyte exchange transfusion as an adjunct to treatment of severely ill patients can be life-saving in selected cases [168], it requires more research, since there has not yet been a prospective clinical study of outcomes of exchange transfusion combined with antimicrobial agents, compared with antimicrobial agents alone.

#### **9. Conclusions**

The spread of infectious diseases among people and animals is a worldwide challenge. The One Health approach provides the opportunity to systematically and comprehensively address emerging zoonoses such as human babesiosis in order to increase awareness of the risk of infection and improve precise diagnostic and seroprevalence tests and treatment protocols. Advances in laboratory methodologies are required to increase our knowledge and understanding of the diversity of zoonotic *Babesia* species and the roles that domestic animals, wildlife and tick populations play in their maintenance. Further development of laboratory tools is necessary for babesia research, including molecular characterization of *Babesia* species and in vitro culture, particularly for testing parasite susceptibility to antibabesial drugs, and the development of screening diagnostics that can be used routinely, for example for the protection of the transfusion blood supply. The improvement of patient

care continues to be important, as awareness is raised among health care professionals and the provision of information on disease prevention behavior is considered by local, national and international governmental institutions.

**Author Contributions:** Conceptualization, J.G., K.-P.H. and E.M.; Investigation A.H., J.G., A.Z., K.- P.H. and E.M.; Writing—original draft preparation, A.H., J.G., A.Z. and K.-P.H.; Writing—review and editing, A.H., J.G., A.Z., K.-P.H. and E.M.; Supervision, K.-P.H. and J.G.; Project administration, E.M. and J.G.; Funding, K.-P.H. Acquisition, K.-P.H. All authors have read and agreed to the published version of the manuscript.

**Funding:** Funding has been provided by a grant from the Society for Promoting Quality Assurance in Medical Laboratories (INSTAND, e.V. Düsseldorf) and a grant from the Health Institute Carlos III (PI20CIII/00037 to EM and LGM), Spain.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

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

#### **References**

