Human Babesiosis-Recent Advances and Future Challenges

Abstract

Purpose of review:

As human babesiosis caused by apicomplexan parasites of the Babesia genus is associated with transfusion-transmitted illness and relapsing disease in immunosuppressed populations, it is important to report novel findings relating to parasite biology that may be responsible for such pathology. Blood screening tools recently licensed by the FDA are also described to allow understanding of their impact on keeping the blood supply safe.

Recent findings

Reports of tick-borne cases within new geographical regions such as the Pacific Northwest of the US, through Eastern Europe, and into China are also on the rise. Novel features of the parasite lifecycle that underlie the basis of parasite persistence have recently been characterized. These merit consideration in deployment of both detection, treatment, and mitigation tools like pathogen inactivation technology. The impact of new blood donor screening tests in reducing transfusion transmitted babesiosis is discussed.

Summary:

New Babesia species have been identified globally, suggesting that the epidemiology of this disease is rapidly changing, making it clear that human babesiosis is a serious public health concern that requires close monitoring and effective intervention measures. Unlike other erythrocytic parasites, Babesia exploits unconventional life cycle strategies that permit host cycles of different lengths to ensure survival in hostile environments. With the licensure of new blood screening tests, incidence of transfusion transmission babesiosis has decreased.

INTRODUCTION

Babesia spp are apicomplexan parasites that divide and replicate in the red blood cells of diverse vertebrate hosts (1). Babesiosis in humans is a consequence of a zoonosis from parasite infection in other species and is characterized by an enzootic cycle between a tick vector and the human host. Infected ixodid ticks that feed on competent natural hosts, including rodents and cattle are the vector source of human babesiosis (2). There are multiple routes of human transmission and these include bites from infected ticks or via a blood transfusion with infected blood (3–5), or congenitally during pregnancy (6–9). There are four identified babesia species that cause human infection: B. microti (10), B. divergens (11) B. duncani (12, 13), and B. venatorum (14–17). In this review, we focus on the changing and expanding range of human babesiosis, novel features of the parasite lifecycle that may raise challenges for detection, treatment or pathogen inactivation strategies and new blood screening platforms that have been licensed by the FDA and assess their impact on transfusion-transmission of the parasite.

Changing Epidemiological Landscape of Human Babesiosis:

Only ticks of the Ixodidae family are confirmed vectors of babesia (18). The two-year lifecycle of I. scapularis, which is endemic across most of the eastern states of America and Canada (19–21), has a prominent role in Babesia transmission (22–24). Various tick stages require a blood meal from their rodent hosts, and adults feed primarily on deer as a permanent food source (20, 22, 24) (25). Both the geographic range of ticks carrying Babesia microti and the incidence of babesiosis have increased significantly over the last 20 years, primarily in the United States (26). B. microti is currently endemic within the Midwest states of Minnesota and Wisconsin and the Northeastern corridor of New Jersey, New York, Connecticut, Massachusetts, and Rhode Island, where its main host, the white-footed mouse, (Peromyscus leucopus) is prevalent (27). Further, a recent incident of B. microti infection in Canada (28) and new cases reported in Pennsylvania (29) shows that transmission is clearly expanding. There have been cases of B. duncani or B. duncani–type organisms in healthy individuals reported in the NW USA (13, 30, 31). Isolated and severe cases of B. divergens–like infections have been reported in asplenic individuals from Missouri (32), Kentucky (33), and Washington State (34). B. divergens is the main pathogen of human babesiosis in Europe (35–37), with the majority of cases being reported in the British Isles and France (38). However, a case of B. divergens in Norway (39), a case of B. microti in Germany (40), coupled with the detection of B. microti in two asymptomatic individuals in Poland (41), and B. venatorum infections reported in Germany (16), Austria and Italy (17) shows again that these pathogens are not absolutely segregated geographically. B. divergens–like infections have been reported in the Canary Islands (20), and other, as-yet uncharacterized babesia species, have been reported in Egypt, Mozambique, South Africa (20, 34, 42). Recent reports of B. venatorum from regions within the People’s Republic of China (14, 15), a pathogen phylogenetically related to B. divergens, and B. microti from south-west China and along the border with Myanmar (43–46), highlight not only the emergence of human babesiosis into new geographical areas,. B. microti–like organisms have been reported in Taiwan (47, 48), Japan (49), South Korea, (50), and a definitive case of B. microti was identified in Australia (51–54).

Parasite lifecycle in humans:

When Babesia spp. sporozoites are first injected via a tick bite into the human host, they invade the host RBCs as their specific target host cell. Invasion occurs using multiple complex interactions between parasite proteins and the host cell surface, all of which are not fully identified and characterized yet (55–62). Once inside the RBC, the parasite begins a cycle of maturation and growth exhibited by intense intra-cellular proliferation. The early stage of the cycle is identical to Plasmodium spp., and both parasites appear as ring stages. Babesia replication occurs by budding (fission), giving rise to a parasite form commonly called the “figure eight” form. Division may occur again, giving ride to the tetrad form know as a “Maltese Cross” (57). These lifeforms are unique to Babesia spp. and are the basis of definitive diagnosis by microscopy, especially if Plasmodium spp. are also suspected. Resulting daughter merozoites egress from the RBCs, lysing it in the process, and seek new, uninfected RBCs to invade, perpetuating the intracellular cycle of infection. Parasite egress is dictated by environmental conditions in terms of both the timing of this critical phase of the life cycle as well as the number of merozoites released from an infected RBC(63).

Host Cell Invasion:

Although invasion of red blood cells (RBCs) is one of the critical points in the lifecycle of Babesia (57), much is yet to be learnt. The exact number of sporozoites that are introduced by the tick bite into the bloodstream of the human host is still unclear making it difficult to assess the relative efficiency of RBC invasion. However, based on in vitro observations using a B. divergens model, not all parasites are successful invaders. The parasite appears to control the number of invaded RBCs perhaps as a way to protect the host and ensure the host survival from the effects of a massive hemolysis that could occur if a high proportion of RBCs are invaded (63). Apicomplexan parasites derive their name from the presence of a unique apical complex that is composed of organelles that play a key role in invasion. They include the micronemes, rhoptries, and dense granules(57). It has been shown that the invasion process is extremely rapid and of the order of seconds. Despite this, the B. divergens merozoites are also extremely hardy and can successfully invade RBCS even minutes after being extracellular, unlike the more fragile malaria merozoites (64). A recent study showed that the invasion time recorded for each free merozoite was different and was completed on average in 72.2 s and the shortest and longest time taken for merozoites to invade the erythrocyte was 38 s and 126 s respectively. Unlike Plasmodium, babesia does not invade other host cells. This specificity implies the presence of receptor(s) on the erythrocyte that is recognized by a complementary molecule(s) on the parasite. Human RBCs that have been treated with different proteolytic enzymes still support parasite invasion, indicating that it has multiple routes of entry into the RBC (56). Although the human RBC has only recently become a host for babesia, the parasite has already established multiple routes of entry. However, babesiosis is a zoonosis and has had to adapt to RBCs from multiple species that possess red cells of different molecular composition, so this ability to successfully invade RBCS that have been stripped of receptors may not come as a total surprise. The parasite thus has a survival advantage when faced with polymorphism of RBC receptor molecules and would still be able to invade and propagate within the RBC. Although this host cell polymorphism should theoretically be matched with a molecular repertoire of parasite ligands that can bind to these multiple host surface receptors, there has been no successful identification yet of cognate receptor-ligand pairs. Recently, a study focused on the genome and transcriptome of the asexual erythrocytic forms of B. divergens was used to identify genes putatively involved in invasion, gliding motility, moving junction formation and egress, providing new insights into the molecular mechanisms of these processes necessary for B. divergens to survive and propagate during its life cycle (65).Through this functional and comparative genomic approach, the authors have identified genes that function as key molecular players in the invasion and egress processes (65).

Babesia exhibits a Flexible Intra-erythrocytic Life Cycle

In a 24 h period, some of the infected RBCs hosting single parasites transform into powerhouses of intense parasitic proliferation, while remaining protected within the same host cells, thus yielding overall parasitemia levels that have not changed dramatically from initial levels. This allows Babesia to build a complex population structure composed of differentially infected RBCs, bestowing the ability to respond promptly to changing external conditions. The advent of synchronization methods for B. divergens has allowed a closer look at the life cycle features of the parasite (63). Unlike other Apicomplexan parasites, B. divergens exhibits a developmental plasticity that affords it multiple options. At high parasitemia, the parasite appears to prolong the RBC cycle, whereas at lower parasitemia, egress of the RBC is chosen to invade new red cells and increase population numbers. Because of the complex morphological presentation of the parasites inside the RBC, the precise life cycle of the parasite has been hard to define and conflicting measures have been reported. Historically, an 8 h lifecycle was described for the parasite, although no clear experimental reasoning had been laid out (66). A 4h life cycle has also been proposed for B. divergens, within the RBC, based on when the first events of egress are noted (67). Other reports have confirmed that 4–5h is an accurate representation of the shortest RBC cycle of the parasite (63). However, there seem to be multiple developmental options available to the asexual Babesia parasite in the RBC with a sequential progression of the seven morphological or pleiomorphic forms of B. divergens seen in culture (Figure 1). It appears that parasite proliferation and differentiation are maintained through strict controls that ensure specific proportions of the different sub populations of parasite infected cells. The asexual cycle can thus be defined as the time required for all pleiotropic forms of the parasite to appear (Figure 1), marked by clear periods of parasite egress followed by new host cell invasion. Using this criterion, we document that 24h is the time needed for the parasite population to complete these events (63). Thus, the flexibility of this phase of the B. divergens lifecycle allows for parasite persistence, depending on specific developmental choices that it adopts. Only merozoites can egress followed by invasion to result in rise in parasitemia. However, these infective units can arise from multiple parasite reservoirs like the paired figure (Fig 1B)or the Maltese Cross (Figure 1C) or two paired figures (Figure 1 E) or the multiple parasites populations (Figure 1F) (63) and this appears to be controlled by the need for population expansion. If there is an urgent need for culture expansion, egress will occur at the two celled stage. If there is a paucity of either host cells or nutrition, double trophozoites develop into double paired figures and paired figures develop into Maltese Cross formation. Parasite populations can resume the pattern of “exponential expansion” once optimum conditions return. A recent review discusses these survival strategies that B. divergens adopts during its intraerythrocytic development to persist and potentially cause asymptomatic disease and reservoir of parasites in the population (68).

FIGURE 1:

Pleiomorphic Forms of Babesia divergens as seen in vitro culture:

A: Ring B: Figure 8 C: Maltese Cross D: Parasite forms ready to Egress

E: Two Figure 8s F: Multiple Parasites

Clinical disease:

Babesia infections vary greatly in their presentation, and are dependent on the infecting parasite species, the age of the host together with its immune competency. Generally, the burden of disease pathology is associated with age, with severe babesiosis presenting in newborns (6–9), or in older adults (38, 69, 70). However, subjects of any age that are immunocompromised, particularly those who are asplenic, are at greater risk from presenting with severe, acute disease, compared to healthy immune competent individuals. B. microti infections in healthy individuals are usually clinically silent and asymptomatic, with very low, almost undetectable levels of parasites (38, 71). Although the long-term effects of circulating parasites are not well understood, the greatest risk of asymptomatic infection is the ability to donate blood and contribute to transfusion-transmitted babesiosis. Mild disease caused by B. microti usually presents with intermittent fever and weakness, often accompanied by chills, sweats, headache, anorexia and myalgia. In patients that were hospitalized with severe B. microti infection, death occurred in about 10% of cases (72, 73). B. divergens infections are much less common than B. microti, (24, 36, 38, 74, 75), but cases present with much more severe pathology, and hemoglobinuria as the presenting symptom. Jaundice due to hemolysis, vomiting and diarrhea are often present, and the toxins and anoxia, resulting from the hemolysis and the host immunological response, may cause respiratory, cardiac, renal, or hepatic failure (35, 75). As such, B. divergens infections require immediate treatment and are treated as medical emergencies.

Transfusion-Transmitted Babesiosis:

Transmission of babesia through blood transfusion has been considered a serious public health threat. Historically, donors living in babesia-endemic areas were prevented from giving blood if they had a history of babesiosis. Because most infections are asymptomatic and not diagnosed, this is not a viable strategy (76–78). As asymptomatic individuals can harbor parasites for extended periods of time, they are able to contribute to the donor pool at any time, not just during the seasons associated with tick-borne infections, thus explaining the incidence of TTB cases year round. Laboratory screening of blood donors for babesia exposure is the optimal preventative measure. Serological and molecular donor-screening assays have been developed and clinical trials have documented reduced TTB in endemic areas (79). Because 0f the increasing risk of TTB, the FDA issued recommendations in May 2019 for reducing risk of transfusion transmission. These protocols include screening donated blood using a licensed antibody and nucleic acid amplification testing (NAT) and/or using pathogen elimination/reduction protocols. These recommendations specifically pertain to areas of babesia endemicity (Connecticut, Delaware, Maine, Maryland, Massachusetts, Minnesota, New Hampshire, New Jersey, New York, Pennsylvania, Rhode Island, Vermont, Virginia, Wisconsin, and Washington, DC) (80).. In other states of the US, a donor questionnaire relating to tick and babesia exposure is recommended to guide donor deferral. Although tests that combined detection of antibody to babesia together with a PCR-based strategy to detect parasite DNA was licensed by the FDA licensure in 2018, this combined assay is not commercially available and not widely used. However, many blood centers have been using a combination of screening for B. microti antibody and B. microti DNA separately and have shown that they can identify and successfully eliminate infectious threats from the blood supply (78, 81). Such methods could thus be used in selected endemic regions to decrease the risk of transfusion-transmitted infections (82). At present, blood donor screening is being performed by investigational NAT which amplify highly repeated babesia RNA sequences in lysed donor whole blood. The sensitivity of the test is very high and can detect 2–3 parasites per milliliter of blood. This is the theoretical infectious dose that a transfused recipient may receive and thus meets the sensitivity requirements of potential testing. Work has also progressed on the characterization of antigens that could form the basis of a serological screen. A library of recombinant B. microti cell surface and secreted antigens was constructed and validated as a valuable resource that could contribute to the development of a serological diagnostic test, vaccines, and elucidate the molecular basis of host-parasite interactions (83). Recently, a genome wide screening for immuno-dominant B. microti antigens has led to the discovery of 56 novel antigens that are attractive diagnostic and vaccine targets. Availability of these antigens also paves the way to develop multiantigen-based B. microti detection assays that would offset the potential sensitivity loss due to geographical antigenic polymorphism and antigenic drift over time (84).

Pathogen Inactivation Strategies

Pathogen reduction processes are another active area of research and represent a proactive approach to both recognized and emerging pathogens including babesia spp. (85). The greatest hurdle to overcome is inactivating the parasite inside the red cell whilst ensuring the absolute competency of the RBCs. These technologies have become significantly more reliable in maintaining red cell integrity whilst making headway in preventing blood-borne pathogens from being transmitted through donated blood. The significant advantage of these technologies is they are being designed as ‘in bag’ treatments that can be ubiquitously applied to all units, without significantly increasing the processing steps between donor and recipient, thus keeping processing costs to a minimum whilst maximizing the safety of the donor pool. Pathogen inactivation methods have been used successfully to reduce B. microti parasitemia in a hamster transfusion model (86, 87). However, studies also show that even a small number of infectious parasites that persist through adverse conditions can lead to large populations of parasites in a relatively short period of time (55). Such persistence strategies by the parasite will have to be addressed before any pathogen inactivation protocols are deemed a success.

Conclusions

Babesia spp are tick-borne pathogen of significant global medical importance as it is associated with transfusion-transmission and relapsing disease in immunosuppressed patients. The clinical epidemiology of human babesiosis appears to be changing and this increased awareness will improve the management of local incidence of disease. Advances in our understanding of parasite biology in the host cell will aid development of new mitigation strategies. The testing and treatment of donor blood is a significant step forward in protecting the blood supply and limiting recipients to the risk of transfusion-transmitted babesiosis, yet more assessment needs to be done to ensure the parasites are completely inactivated without compromising the integrity of the blood components. However, as clinical information becomes more readily available, there are significant gaps in our understanding of the basic biology of these parasites which makes uncovering critical aspects of the parasite biology urgent and much warranted.

KEY POINTS.

• The geographic range of ticks carrying Babesia and the incidence of babesiosis have increased significantly over the last 20 years, primarily in the United States.

• Recent advances in methodology have allowed the refinement of biological parameters of the parasite life cycle and suggest reasons for resistance of parasite to traditional pathogen reduction protocols

• To eliminate the risk of transfusion transmitted babesiosis, the FDA has issued recommendations for testing donor blood in babesia endemic areas of the US.