Transfusion-transmitted Babesia spp.: a changing landscape of epidemiology, regulation, and risk mitigation

ABSTRACT

Babesia spp. are tick-borne parasites with a global distribution and diversity of vertebrate hosts. Over the next several decades, climate change is expected to impact humans, vectors, and vertebrate hosts and change the epidemiology of Babesia. Although humans are dead-end hosts for tick-transmitted Babesia, human-to-human transmission of Babesia spp. from transfusion of red blood cells and whole blood-derived platelet concentrates has been reported. In most patients, transfusion-transmitted Babesia (TTB) results in a moderate-to-severe illness. Currently, in North America, most cases of TTB have been described in the United States. TTB cases outside North America are rare, but case numbers may change over time with increased recognition of babesiosis and as the epidemiology of Babesia is impacted by climate change. Therefore, TTB is a concern of microbiologists working in blood operator settings, as well as in clinical settings where transfusion occurs. Microbiologists play an important role in deploying blood donor screening assays in Babesia endemic regions, identifying changing risks for Babesia in non-endemic areas, investigating recipients of blood products for TTB, and drafting TTB policies and guidelines. In this review, we provide an overview of the clinical presentation and epidemiology of TTB. We identify approaches and technologies to reduce the risk of collecting blood products from Babesia-infected donors and describe how investigations of TTB are undertaken. We also describe how microbiologists in Babesia non-endemic regions can assess for changing risks of TTB and decide when to focus on laboratory-test-based approaches or pathogen reduction to reduce TTB risk.

INTRODUCTION

Babesia spp. are intraerythrocytic protozoan parasites of animals and humans that cause babesiosis. Babesia microorganisms were first described in 1888 by Victor Babes who sought the cause of red water fever in Eastern European cattle that caused extensive mortality (1). While Babes identified the parasite that would subsequently bear his name, he was unable to decipher the transmitting agent. Ticks were identified as the vector for Babesia by Theobold Smith and F. L. Kilbourne while investigating Texas cattle fever (2). Their seminal paper, published in 1893, represented the first description of vector-borne disease (3). It was not until 1957 that the first human case of babesiosis was described in a splenectomized herdsman residing near present-day Zagreb, Croatia (4). The probable etiologic agent was initially identified as B. bovis but was likely B. divergens (5). Decades later, the first case of transfusion-transmitted Babesia (TTB) was reported in 1980 (Boston, MA) when a 70-year-old patient was infected with B. microti following multiple platelet transfusions (6).

Since the early 1980s, the epidemiology of Babesia and the medical community’s response has rapidly evolved. Throughout the ensuing decades, the number of TTB cases increased in frequency, albeit almost exclusively in the United States. Concerns among transfusion medicine practitioners led to epidemiologic studies to identify the areas of risk and the frequency of transmission (7, 8). Two risk areas were identified, the Northeast and Upper Midwest, where Babesia was transmitted by Ixodes scapularis ticks, the same tick species that transmits Lyme disease (9). Over time, the risk areas have expanded due to evolving ecosystems, especially a marked increase in the number and geographic range of white-tailed deer that markedly amplify tick numbers (10). More recently, climate change is also thought to contribute to the parasite’s expanding range (11, 12). In response to an emerging blood safety risk, a variety of mitigation efforts were implemented. These eventually lead to Food and Drug Administration (FDA) licensure of sensitive and specific nucleic acid test (NAT) assays that have proven effective at reducing or eliminating transmission of Babesia in tested blood products (13).

The objective of this review is to assess the current status of risk posed by Babesia spp. to transfusion medicine. This review highlights recent insights regarding the epidemiology of Babesia spp. in the United States and in other regions where they represent an emerging threat. We also examine contemporary management practices at transfusion centers, including donor and TTB case management. Lastly, the review describes mitigation strategies designed to reduce TTB, their effectiveness, and considerations for their implementation depending on perceived risk.

The taxonomy and survival of Babesia in nature

The genus Babesia is taxonomically located within Superkingdom: Eukaryota, Clade: Alveolata, Phylum: Apiocomplexa, Class: Aconoidasida, Order: Piroplasmida, and Family: Babesiidae (14). Species and strains of Babesia that have been shown to cause human disease include B. crassa-like (15 – 18), B. divergens (19, 20), B. divergens-like (21), B. duncani (22), B. microti (23), B. microti-like (24), B. motasi-like (25), and B. venatorum (11, 16, 26). A much larger group of over 100 Babesia species infect vertebrates but have not been linked to human disease (14, 16, 27, 28). Babesia spp. are global pathogens with most human cases reported in Asia, Europe, and North America (Table 1). A recent review also describes sporadic cases in Australia and Africa and provides a world map depicting the major areas of Babesia spp. transmission (16). Whole-genome sequencing data are available for B. divergens (29, 30), B. duncani (31, 32), B. microti (33 – 37), and B. motasi (38). B. microti and B. duncani have the smallest genomes (Table 1). Using 18S rRNA molecular phylogeny (39), Babesia species can be clustered into two groupings, Babesia sensu stricto (B. crassa, B. divergens, B. motasi, and B. venatorum) and Babesia sensu lato (B. duncani and B. microti) (40). A separate clade division system with a further division system has been reviewed elsewhere. (Table 1) (27, 28). Genome sequencing and comparative genomics afford novel insights into the pathophysiology, evolution, and parasite-host interactions of different Babesia species and lineages (38, 41). These can be also used to develop new diagnostic tools for the clinical microbiology laboratory (42).

TABLE 1.

Global distribution and biological characteristics of Babesia species implicated with human disease

Babesia-type species	B. crassa-like	B. divergens	B. duncani	B. microti	B. motasi	B. venatorum	References
Clade as designated in Schnittger et al.	VI	VI	II (Western group)	I (B. microti-like group)	VI	VI	(27, 28)
Classic taxonomy	Babesia sensu stricto	Babesia sensu stricto	Babesia sensu lato	Babesia sensu lato	Babesia sensu stricto	Babesia sensu stricto	(40)
Most common location of human disease	Asia	Europe USA	Western North America	North-eastern and midwestern USA/Canada Asia	Asia	Europe Asia	(16, 25, 43)
Suspect or confirmed main vectors	Haemaphysalis concinna, I. persulcatus	I. ricinus	Dermacentor albipictus	Haemaphysalis longicornis, I. scapularis (I, dammini), I. ricinus, I. persulcatus, I. ovatus, I. trianguliceps, I. ricinus, Rhipicephalus haemaphysaloides	Haemaphysalis longicornis (possible), Haemaphysalis punctata, Rhipicephalus bursa, Dermacentor reticulatus	I. ricinus (Europe)	(18, 25, 39, 44 – 46)
Transovarial transmission in ticks	Yes	Yes	Not documented	Not documented	Yes	Yes	(40, 47 – 49)
Non-human animal reservoir hosts	Dogs, sheep, cattle	Cattle	Mule deer	Small mammals (e.g., mice, rodents)	Sheep, goat	Sheep, deer	(18, 39, 48, 50 – 52)
Whole-genome sequencing information available	No	Yes	Yes	Yes	Yes	No	(29 – 38)
Genome size (Mbp)	− a	9.7	7.9	6.4	14.5	−
GC%	−	42.5	37.7	36.2	46.9	−
Coding gene numbers	−	3741	3759	3573	−	−
Gene density (genes/Mb)	−	385.7	475.8	558.3	−	−

^a –, unavailable.

The life cycle of Babesia consists of alternate residence within a tick vector and a mammalian reservoir host, as shown for Babesia microti, the most common species that infects humans. While B. microti has been recovered from several species of birds, the avian role in maintaining this parasite is not clear (53). Tick vectors that transmit other Babesia species which infect humans include I. ricinus, I. persulcatus, Dermacentor albopictus, Haemophysalis concinna, and H. longicornis. Animal reservoirs include mice, shrews, rabbits, domestic and wild ungulates, monkeys, and birds (54). (Table 1) (44, 55, 56).

A more in-depth review of the life cycle of Babesia in tick and vertebrate hosts is described elsewhere (39). Humans can interact with hard-bodied ticks at any tick stage (e.g., larva, nymph, and adult), but the nymphal stage most commonly transmits Babesia to humans (53, 57 – 59). A tick bite results in infection through the transmission of sporozoites in the tick saliva. Sporozoites invade erythrocytes and develop into trophozoites which then multiply asexually (merogony). Asexual reproduction leads to the production of both merozoites (infectious to other erythrocytes) and gametocytes.

The survival of Babesia in blood components

TTB has been linked to the transfusion of red blood cells (RBCs), frozen deglycerolized RBCs, and whole blood-derived platelet concentrates (9, 60). No confirmed cases of TTB have been ascribed to apheresis platelets, cryoprecipitate, or frozen plasma products (61). In the absence of a cryopreservative, Babesia parasites do not retain viability following a freeze-thaw cycle (62). Within red blood cells, experiments undertaken in blood collection tubes (and not gas-permeable bags) suggest that B. microti is able to survive at 4°C for at least 21 days (63). In culture-based temperature experiments, B. divergens was introduced into leukoreduced red blood cells stored in blood bags for up to 31 days. This survival probably exists in real-world settings as described in a TTB case caused by transfusion of a 35-day-old red cell unit (64). The number of viable parasites that are needed to cause TTB is low (65). In one study, an inoculum of 10–100 B. microti parasitized red blood cells resulted in infection and chronic parasitemia in immunocompetent mice (66). Multiple B. microti parasites per red blood cell may also enhance the infectivity when there are a low number of infected cells (67).

Epidemiology of tick-borne babesiosis and TTB

In the United States, the most common Babesia spp. infecting humans is B. microti, transmitted by I. scapularis to small mammal reservoirs such as the white-footed mouse (Peromyscus leucopus) (Table 1). While B. microti has been recovered from several species of birds, the avian role in maintaining this parasite is not clear (53). Other Babesia species that infect humans in the United States include B. duncani with a Dermacentor albopictus tick vector; and B. divergens-like pathogen, possibly transmitted by I. dentatus. In Europe, Babesia species that infect humans include B. divergens with a I. ricinus tick vector and large ungulates (bovine, cervid) as reservoir hosts (68); B. venatorum with an I. ricinus tick vector and roe deer reservoir; B. crassa-like agent, possibly vectored by Haemaphysalis concinna; and B. microti with a I. ricinus tick vector. In China, Babesia spp. that infect humans include Babesia crassa-like agent with I. persulcatus and Haemaphysalis concinna as tick vectors, and goats and sheep as probable reservoir hosts; B. divergens; B. microti; and B. venatorum (16, 18, 44) (Table 1). In sum, implicated vectors of human Babesia spp. pathogens include Ixodes species, Dermacentor species, and Haemophysalis species, while implicated animal reservoirs include mice, shrews, rabbits, domestic and wild ungulates, monkeys, and birds (54) (44, 55, 56) (Table 1).

Globally, there has been a marked increase in cases of babesiosis due to tick and transfusion transmission of which the overwhelming majority (~99.5%) are tick transmitted. In the United States, between 2011 and 2019, 16,456 babesiosis cases were reported to the Centers for Disease Control and Prevention (CDC) from 37 states with 98.2% of the cases reported from 10 states. Among the 10 states, there was a trend of increasing incidence (median incidence 117.5%, range −28.9 to 1601.8%) between 2011 and 2019 (69). This same report also indicated that over that time, Maine, New Hampshire, and Vermont developed levels of endemic tick-borne transmission that are similar to those of other high-incidence states (69).

Almost all cases of tick-borne babesiosis occur between May and September when ticks are actively seeking a blood meal and people are often outdoors. By contrast, TTB occurs throughout the year. Only a little over half of the TTB cases occur during the tick transmission season (9). About a fifth of all Babesia infections are asymptomatic in adults (70). Asymptomatic infection can persist for months in humans (even after antibiotic therapy), and individuals may donate infected blood throughout the year without realizing that they are infected (9, 71). The geographic distribution of tick-borne babesiosis and TTB is similar. Most cases are reported in the northeastern United States (72). However, 17% of cases of TTB are reported outside the Northeast as compared to 8% of cases of tick-borne disease (Table 2). In the United States, cases of TTB have historically been reported in areas that were not regarded as endemic for Babesia. This is ascribed to the frequent transportation of blood across US state lines, whereby blood that was collected in an endemic area might be transfused in a non-endemic area. Furthermore, blood donors living in non-endemic areas may travel to endemic areas where they become infected and then donate blood after returning home (73, 74). Tick-transmitted infection is more common in males, and this has been postulated as a result of differences in activities that increase the risk of exposure to ticks (e.g., hunting, hiking) (75).

TABLE 2.

A clinical comparison of tick-transmitted vs TTB

Category	Tick transmitted (% cases)	Transfusion transmitted (% cases)	References
Cases			(9, 73)
Location
New England	53	40
Mid-Atlantic	39	43
Midwest	7	12
South	0.5	2
West	0.5	3
Total	100 a	100 b
Age distribution			(9, 75)
1–50 years	22	32
51–90 years	78	68
Total	100 c	100 d
Gender			(9, 75)
Male	65	50
Female	35	50
Total	100 e	100 f
Race g
White	59	-	(75)
Asian/Pacific Islander	3
Black	2
Other	2
Unknown	34
Total	100 h
Symptoms			(8, 70, 76)
Fever	89	74
Fatigue	82	48
Sweats	58	13
Headache	44	0
Myalgia	39	9
Chills	68	35
Anorexia	53	39
Cough	27	17
No symptoms	20	13
Total	100 i ()	100 j
Complications
Cardiac (CHF)	11	11	(76 – 78)
Pulmonary distress (ARDS)	10	33
Kidney (renal failure)	5	22
DIC	18	22
Shock	4	11
Total	100 k	100 l
Death	7	19	(74, 77, 78)
Total	100 m	100 n

^a 16,397 cases.

^b 141 cases.

^c 7173 cases.

^d 134 cases.

^e 7,173 cases.

^f 134 cases.

^g Analysis of race analysis was not available for transfusion-transmitted babesiosis.

^h 7,612 cases.

ⁱ 249 inpatient cases.

^j 23 inpatient cases.

^k 173 cases.

^l 9 cases.

^m 173 cases.

ⁿ 154 cases.

Most TTB cases in the United States have been reported in the northeastern United States where B. microti is endemic (Table 2). The earliest published report on TTB was in 1982 (6, 79). By the 2000s, blood donor studies demonstrated a high seroprevalence for B. microti in endemic areas [Connecticut (112.4 per 10,000 donations), Massachusetts (140.4 per 10,0000)] (80). Antibody positivity has also been identified in blood donors from areas adjacent to highly endemic areas, as well as those geographically distant from endemic areas (81, 82). The potential risk of TTB in non-endemic states is illustrated by reports of TTB in non-endemic states such as Texas and California, which were ascribed to donors who had spent time in the northeastern United States prior to donation (83, 84). The risk of TTB is similar between men and women (9).

The breadth of studies used to characterize the risk of TTB in the United States and Canada is lacking in other settings. To date, there have been a total of two case reports of TTB (85, 86), three blood donor serosurveys (87 – 89), and one blood donor molecular survey outside of North America (Table 3) (90). Furthermore, these few studies are heterogenous in their design, sample sizes, and locations. Collectively, they offer only gross under-representation by population, demography, and geography. In some cases, the assays that were employed had not been validated for the specific populations in which the studies were undertaken, further detracting from their findings (87).

TABLE 3.

Cases of transfusion-transmitted babesiosis outside North America

Location(s) and year(s)	Study design	Overview	Reference
Japan 1998–1999	Case report	A 40-year-old male investigated for fever and hemolysis 1 month after transfusion for gastric bleeding. • Parasites visible on blood smear • B. microti infection confirmed by PCR • Donor implicated	(85)
Germany 2006	Case report	A 42-year-old female investigated with acute myeloid leukemia presented with fever and chest pain after her first cycle of chemotherapy. The patient had received 45 platelet concentrates and 11 packed red blood cells in the 2 months prior to diagnosis of babesiosis. • Parasites visible on blood smear • B. microti infection confirmed by PCR • Donor implicated; borderline reactivity B. microti (IgG titer 1:32)	(86)
New South Wales and Queensland, Australia 2012–2013	Pilot serosurvey	Retrospective IFA screening of blood donor plasma samples for B. microti IgG; reactive samples were tested for B. microti IgG and IgM by immunoblot and B. microti DNA by PCR • None (0%) were confirmed as seropositive • Five initial reactive donors were not confirmed on repeat/confirmatory testing	(88)
North and East Tyrol, Austria Year not stated	Pilot serosurvey	Retrospective IFA screening for B. divergens and B. microti (cutoff titer 128) • 21/988 (2.1%) B. divergens IgG positive •5/988 (0.6%) B. microti IgG positive	(89)
Heilongjiang Province, China 2016	Pilot serosurvey	Retrospective IFA screening of blood donor samples (n = 1,000) for B. microti antibodies •888 whole blood and 112 platelet donors •13/1,000 (1.3%) seroreactive • 0.8% at a titer of 64 and 0.05% at a titer of 128	(87)
Tyrol, Austria	Molecular survey	Prospective molecular testing of blood donors at regional, mobile blood collection drives in Tyrol, Austria (27 May to 4 October 2021). Testing was conducted with a commercial PCR assay approved for blood donor screening; the assay can detect the four primary species causing human babesiosis (i.e., B. microti, B. divergens, B. duncani, and B. venatorum). • None (0%) of 7,972 donors were PCR positive	(90)

Although Babesia spp. are pathogens that are primarily of concern to blood operators in North America, the parasites have rarely been identified in blood donors outside of North America. The rarely performed studies that have been undertaken on blood donors suggest that the risk of TTB—at least in those highly selected areas that were studied—is low or even absent (88, 90). That would support the current policy of risk-based deferral rather than laboratory-based screening. At the time of this writing, the United States is the only country that performs routine donor testing for Babesia (91). Case reports of TTB—albeit rare (85, 86)— coupled with the global emergence of babesiosis (16), offer plausibility of transfusion transmission outside of North America, suggesting that cases of TTB may be occurring and are undetected. Babesia is globally ubiquitous, and B. microti accounts for almost all cases of TTB (92 – 95). Most other Babesia spp. that infect humans (B. crassa-like (15 – 18), B. divergens, B. divergens-like, B. duncani (19, 20), B. motasi-like (25), and B. venatorum (96)) have not been studied in blood donors or transfusion recipients. In short, there are insufficient data to draw meaningful conclusions regarding the risk of TTB outside of North America. The risk may well be low, but the data are insufficient for solid conclusions.

Climate change, changing ecosystems, and the potential for global changes in Babesia epidemiology

There are myriad factors that impact the distribution and risk of Babesia with a complex interplay of the human-vector-reservoir triad (11, 12, 97 – 100). Climate change and the associated increase in temperature and humidity favor the survival and activity of ticks and their reservoir hosts in northern climates. This has resulted in the expansion of B. microti-infected I. scapularis ticks into Canada, albeit with only a few autochthonous cases reported (97).

Other transfusion-transmissible bacterial and viral tickborne agents can be transmitted by the same tick vectors (I. ricinus complex, Haemaphysalis spp.) as Babesia spp. Currently, Anaplasma phagocytophilum is the most frequent transfusion-transmitted tickborne agent after Babesia spp. (Table 4). Changes in the regional epidemiology of these pathogens may portend a change in Babesia epidemiology and subsequent risk of collecting a blood donation from Babesia-infected blood donors (101 – 105). Recent studies have shown that B. microti-infected white-footed mice (P. leucopus) are capable of vertically transmitting B. microti to their offspring (106). This non-vector-mediated pathway may further contribute to the emergence and geographic spread of B. microti. Similarly, in Europe, Ixodes spp.-infected ticks are expected to expand northward following warmer and more humid conditions while their distribution may contract in southern drier areas (11). The temperature changes will not just impact tick distribution but also alter seasonal feeding behavior such that nymphal ticks may infect small mammals at the same time larval ticks are acquiring the infection, potentially resulting in B. microti “hot spots” (107). Human influence on the environment also creates new risks for B. microti transmission. The re-colonization of the northeastern United States by white-tailed deer (Odocoileus virginianus) following the retreat of land-clearing farming practices is a widely accepted cause for the rise of tickborne infections because deer are the preferred host of adult I. scapularis and significantly increase the number of ticks wherever they are found (108). Conversely, the reduction of local deer populations has resulted in a marked decrease in I. scapularis number and Lyme disease cases (109). Introduction of ticks by birds or commercial transport increases the risk of babesiosis and other tick-borne diseases. In Europe, B. venatorum is typically associated with roe deer (Capreolus capreolus). An increase in roe deer populations with the associated increase in the I. ricinus tick population increases the risk for babesiosis, especially for hunters and others spending time in wooded areas (110). While B. venatorum has not been found in wild deer in the United Kingdom, one study detected this pathogen in sheep, which was likely introduced through avian transport of the I. ricinus tick vector (50, 111).

TABLE 4.

Transfusion-transmitted pathogens that share common tick vectors with Babesia species

Pathogens	Common vector tick	Region	Transfusion-transmission notes	References
Anaplasma phagocytophilum	I. persulcatus I. ricinus I. scapularis	North America, Europe, Asia	12 TT cases described	(112)
Ehrlichia ewingii	I. persulcatus I. scapularis	USA, Mexico, Europe, Asia	1 case	(112, 113)
Powassan virus	I. scapularis	USA and Canada	1 possible case in USA	(114, 115)
Tick-borne encephalitis virus (TBEV)	Dermacentor reticulatus Haemaphysalis concinna H. punctata I. persulcatus I. ricinus	Europe and Asia	2 cases described in Finland	(116 – 120)

U.S. surveillance case definition for babesiosis

In the United States, criteria have been established by the Centers for Disease Control and Prevention to define cases of babesiosis in humans to facilitate epidemiological surveillance (121). These case definitions are important tools as analysis of data from 2011 to 2019 suggests that the number of babesiosis endemic states in the United States has increased with the addition of Maine, New Hampshire, and Vermont. This analysis also called for increased vigilance for babesiosis in endemic states and in states that border endemic states (69). Cases are defined as “Suspected,” “Probable,” or “Confirmed” based on clinical, laboratory, and epidemiological data. Given the objectives of public surveillance, the case definitions are not intended to be used by clinicians to aid clinical diagnosis or patient management. CDC case definitions include the potential for TTB. A linked blood donor would have “donated at least one of the RBC or platelet components that was transfused into the recipient” and “the plausibility that this blood component was the source of infection in the recipient is considered equal to or greater than that of blood from other involved donors (more than one plausible donor may be linked to the same recipient).” A linked recipient would have “received one or more red blood cell or platelet transfusions within 1 year before the collection date of a specimen with laboratory evidence of Babesia infection” and “at least one of these transfused blood components was donated by the donor described below,” and “transfusion-associated infection is considered at least as plausible as tickborne transmission” (121).

Clinical comparison of babesiosis acquired through tick transmission and blood transfusion

The incubation period after tick-transmitted babesiosis is 1–4 weeks; by contrast, the incubation after TTB is 1–9 weeks but may be as long as 6 months (9, 70, 122). About a fifth of adults and over a third of children are asymptomatically infected with B. microti following tick bite (71, 72). The severity of the illness ranges from mild to severe. Mild-to-moderate disease is a characteristic for children and immune-intact adults and moderate-to-severe illness for older (>50 years) and immunocompromised patients (70). Patients who acquire Babesia through blood transfusion frequently experience a moderate-to-severe illness, as most of them have comorbid and/or immunocompromising conditions that predispose them to severe disease; however, some cases of asymptomatic TTB have been described (8, 70, 75, 76).

In an analysis of 6,493 reported cases of babesiosis in the United States from 2011 to 2015, fever (84%) was the most common symptom, followed by chills (70%), myalgia (68%), headache (60%), and sweats (55%) (75). The frequency of these symptoms was similar to those of 290 babesiosis out-patients and in-patients who took part in several studies carried out in the northeastern United States (Table 2) (70, 77, 78). Symptoms are similar between tick-borne and TTB cases, but the relative frequency of specific symptoms differs for reasons that remain unclear. The relatively low number of TTB patients from whom symptom data are available may distort the true clinical spectrum encountered in TTB (Table 2) (8, 76).

The same complications of babesiosis have been reported for tick-borne and TTB (Table 2). While the frequencies of complications differ, the number of study subjects in the TTB group was small (Table 2). Most importantly, death was more than twice as likely in those with TTB (19%) than in those with tick-borne transmission (7%) (9, 77, 78). This is ascribed to the high proportion of comorbid risk factors for severe disease among transfusion recipients, while most patients with tick-borne babesiosis are in relatively good general health. In the future, other tools such as administrative and admissions databases may assist researchers in understanding changing demographics of babesiosis, particularly in non-endemic regions (72, 123).

The diagnosis of tick-borne babesiosis and TTB is made by medical history, physical examination, and laboratory testing. Laboratory confirmation of babesiosis consists of identification of Babesia microorganisms on thin blood smears and/or Babesia nucleic acid (70). The standard treatment for both tick-borne babesiosis and TTB is atovaquone and azithromycin, which are administered for 7–10 days (124). A regimen of clindamycin and quinine is an alternative. The 2020 Infectious Diseases Society of America guidelines on the diagnosis and management of babesiosis recommend continuation of antibiotic therapy for at least 6 weeks for highly immunocompromised patients with at least two consecutive weeks of negative blood smears. The guidelines also suggested that step-down therapy to an all-oral antimicrobial regimen is appropriate when symptoms have abated (124). In immunocompromised patients with prolonged courses of antibiotic, the allelic changes at specific gene loci are associated with antimicrobial treatment failure for atovaquone (cytb, cytochrome b), azithromycin [rpl4, azithromycin-binding region of ribosomal protein L4 (RPL4)], and clindamycin (23S rRNA) (125 – 127). Prevention of tick-transmitted babesiosis consists of avoiding tick-infested areas during the transmission season, use of long sleeve shirts and long pants tucked into socks, clothing sprayed or impregnated with permethrin, application of tick repellant to skin (N,N-diethyl-meta-toluamide), and removal of attached ticks with tweezers as transmission generally requires 36–72 hours of tick attachment (128). Screening blood donations prior to transfusion for Babesia is highly effective in reducing TTB (129 – 132).

There has been a significant decline in cases of TTB since the implementation of regional screening of blood donors for Babesia in the United States (130 – 132). Cases of TTB are now rarely reported (133). From June 2012 through September 2014, the American Red Cross conducted a large-scale, investigational product-release screening and donor follow-up program. Donors at selected blood drives in Connecticut, Massachusetts, Minnesota, and Wisconsin were tested using both an arrayed fluorescence immunoassay (AFIA) and a polymerase chain reaction (PCR) assay for B. microti. None of the 75,331 screened donations (0:75,331) were implicated in TTB. By contrast, 14 out of 253,031 (1:18,074) unscreened donations were implicated in cases of TTB. One caveat from the study was that the natural transmission of Babesia could not be completely excluded. Following the 2019 FDA Recommendations for Reducing the Risk of Transfusion-Transmitted Babesiosis Guidance for Industry (13), the American Red Cross in 2020 implemented blood Babesia NAT for all donations in endemic areas of the midwestern and northeastern United States (14 states and Washington DC). Between 5 May 2020 and 31 May 2021, 1,816,669 donations were tested and 365 reactive donations corresponding to 365 donors were identified. From this group, 97% (n = 355) were confirmed with further NAT-positive or an antibody-positive result. The 365 initially reactive donations were collected in every US state except Delaware. The highest proportions of Babesia reactive donors were encountered in the following states: Massachusetts (1:1,865), Connecticut (1:1,881), New Jersey, (1:2,067), Maine (1:3,774), New York (1:5,077), New Hampshire (1:5129), and Pennsylvania (1:5,779). In general, the state in which the donation occurred corresponded to the donor’s state of residence. In some cases, donors donated in a state adjacent to where they resided. For the period under investigation, the American Red Cross received no notification of TTB cases. This contrasts with the period 2010–2020 when a yearly average of 14 TTB cases and 8 positive donors were identified (132). The risk of TTB may not be completely mitigated with donation testing from specific US states. The expanding range of both Babesia and its tick vector includes states that were not included in the list of states in which donation testing was recommended (69). Donors who reside and donate in non-endemic states where there is no mandated donation testing may still become infected with Babesia during travel to endemic regions (133).

Test characteristics of diagnostic and blood operator tools for detecting Babesia infection

A general understanding of the test characteristics of clinical laboratory, reference laboratory, and blood donor screening assays is important when investigating potential TTB.

Blood donor screening: NAT

Two NAT assays detecting DNA or RNA are FDA licensed and available to screen blood donations collected in endemic areas in the United States. These include the Procleix Babesia Assay (transcription-mediated amplification, Grifols Diagnostic Solutions, Barcelona, Spain) (134) and the cobas Babesia (real-time reverse-transcription PCR, Roche Molecular Systems, Inc., Basel, Switzerland) (135). Both assays can detect the four Babesia species primarily associated with human infection (B. microti, B. divergens, B. duncani, and B. venatorum) and can be performed on fully automated platforms. One assay uses transcription-mediated amplification (TMA) for the detection of the 18S rRNA, with an analytical sensitivity of 1.8–3.1 parasite/mL. The other assay is a PCR-targeting ribosomal RNA and DNA, with an analytical sensitivity between 0.5 and 9.6 iRBC/mL, depending on the Babesia species (134, 135).

Clinical and reference testing: blood film microscopy

A diagnosis of babesiosis can be confirmed by microscopic detection of the parasite within red blood cells on Giemsa or Wright-stained thin and thick blood smears (136). However, parasitemia lower than 1% is common in the early stage of infection and in asymptomatic individuals, and low parasitemia Babesia infections can be easily missed in thin smears if at least 300 fields or multiple smears are not examined. Babesia trophozoites resemble P. falciparum ring structures (without pigment) but are generally larger with varied size and shape compared with P. falciparum. Merozoites arranged in a tetrad (‘‘Maltese cross”) are pathognomonic for Babesia spp. but are rarely noted. In sum, the two parasites are morphologically similar, and misdiagnoses have been reported (137). Maintenance of microscopy skills by clinical laboratory staff is important even in the era of NAT (138). In cases where Babesia infection is not considered in the differential diagnosis, microscopy may identify parasites incidentally, thereby helping to guide further laboratory testing and patient management (139). In some settings, when Babesia infection is suspected, microscopy and a history of travel to or residence in an endemic area may assist in diagnosis and initiating patient management in a timely manner (138).

Clinical and reference testing: experimental inoculation

Babesia divergens, B. microti, and B. duncani can be cultivated in gerbils, mice, and hamsters; however, xenodiagnosis is impracticable for clinical diagnosis, given that it is both labor intensive and time-consuming. Similarly, the in vitro cultivation of piroplasms requires sophisticated techniques, is labor intensive and costly, and is reserved for specialized laboratories (140).

Clinical and reference testing: Babesia NAT

In addition to the previously described NAT assays developed for blood donor screening, nucleic acid-based testing performed by PCR is used in the clinical setting. Babesia NAT has played an important role in identifying autochthonous cases in regions in which locally acquired infections were previously unknown (95, 141). Clinical and reference laboratory NAT has also played a role in investigating blood product recipients for TTB (139). These assays usually target the 18S ribosomal ribonucleic acid (rRNA) gene and are both highly sensitive and specific. They also enable species identification. No commercial assay has been licensed yet for diagnostic use, but in-house assays are used mainly in reference laboratories. Real-time PCR assays can detect as low as 1,000–3,000 parasites/mL in whole blood specimens (142 – 145).

Clinical and reference testing: antibody detection assays

Enzyme-linked immunosorbent assay and indirect immunofluorescence assay, including AFIA designed to facilitate high-throughput screening, have been developed for the detection of B. microti-specific antibodies and used to determine B. microti seroprevalence in blood donors in the United States (80 – 82, 134, 146, 147). Serological assays can detect antibodies in 88%–96% of patients with B. microti infection and are a reliable marker of Babesia exposure (146). Serological tests cannot definitively distinguish between current and past infections and may not detect early infections when antibody response is not yet present (134).

Clinical and reference testing: antigen detection assay

Several publications describe other immunoassay formats (e.g., enzyme immunoassays, bead-based assays, or immunoblots) that use various antigens. The use of antigens in human babesiosis diagnostic tests is still under development, and laboratory or commercial antigen-based tests are unavailable. A promising antigen-capture assay targeting B. microti alpha-helical cell surface protein 1 (BmGPI12, also known as BmSA1) using polyclonal and monoclonal antibodies was shown to detect active B. microti infection in 97.1% NAT-positive human serum and plasma (148, 149).

Donor and TTB case management

2019 US recommendations

The US FDA’s May 2019 Babesia Guidance considers babesiosis to be a relevant transfusion-transmitted infection under 21 CFR 630.3(h) (2). The guidance in that document “applies to the collection of blood and blood components, except source plasma (13).” Blood operators must test each donation using a licensed Babesia NAT in Connecticut, Delaware, Maine, Maryland, Massachusetts, Minnesota, New Hampshire, New Jersey, New York, Pennsylvania, Rhode Island, Vermont, Virginia, Wisconsin, and Washington, D.C. or implement pathogen reduction technology for platelets and plasma using an FDA-approved pathogen reduction device effective against Babesia, according to the manufacturer’s instructions for use (13). The FDA guidance also provides information on Babesia-risk deferral approaches in non-endemic regions of the United States. In the event of a donor testing positive with a licensed Babesia NAT, the FDA document provides guidance on how to investigate all cellular blood components collected from that donor in the prior 12 months (13).

Lookbacks: when a blood donor is infected with Babesia

Following the detection of a positive test result for a transmissible disease (either by a clinical laboratory or a blood screening test), a lookback procedure is initiated to investigate the index donor’s prior donations (139, 150). Lookbacks can be multi-jurisdictional and depending on the timing of exposure may involve both Babesia NAT as well as serology (139). Lookback investigations may involve multiple clinical microbiology laboratories in which different Babesia NAT or serology tests with different sensitivities and specificities are employed (139).

TTB case management: tracebacks

When a blood recipient tests positive for a Babesia infection by a clinical laboratory test, the implicated donor(s) associated with the blood components transfused are identified (e.g., traced back) and retested for the appropriate transmissible disease. Depending on available resources, retesting may be carried out by a clinical laboratory assay or blood donor screening assay (150, 151). The goal is to identify any Babesia-infected donors. If a donor is implicated, then that donor’s other donations may be investigated in a related lookback.

Mitigation

Exclusion approaches

Risk-based deferral through questioning a donor regarding a history of babesiosis is largely ineffective, as donors are expected to be healthy (i.e., asymptomatic) thus reflecting a lack of awareness of their being infected with Babesia (60, 152). A history of a tick bite is not included in North American blood donor questionnaires, given that it has been shown to be a poor predictor of exposure (153). In the United States, exclusion approaches may still occur in regions where Babesia is not endemic and mandatory blood donor screening has not been implemented. For example, blood operators in the United States ask donors if they have ever had a positive test result for Babesia, either with a clinical or with a donor screening test (13).

Pathogen reduction technologies

Pathogen reduction (PR) refers to a variety of technologies {e.g., photochemical inactivation (psoralen/ultraviolet light (UV) A, riboflavin/UV light), solvent detergent treatment, nanofiltration} to treat blood products (154). PR with photochemical inactivation has been used successfully to decrease the risk of bacterial contamination of platelets, given the availability of FDA and CE-marked commercial technologies (155). There are still no approved PR approaches for the treatment of red blood cells or whole blood, however, which are the major blood products that are associated with the risk of TTB. Few studies have assessed the efficacy of PR technologies against B. microti (Table 5). In vitro data indicate that four technologies, namely INTERCEPT Blood System for platelets (Cerus Corporation, Concord, California, USA), INTERCEPT Blood System for red blood cells (Cerus Corporation), Mirasol (Terumo BCT, Tokyo, Japan), and THERAFLEX UVC-Platelets (Macopharma, Montreal, Quebec, Canada), effectively reduce B. microti infectious microbial loads in blood components by several orders of magnitude (Table 5).

TABLE 5.

Analysis of original articles providing experimental results on inactivation of B. microti by pathogen reduction technologies

Pathogen reduction technology	Main results	Remarks	Reference
N-(4-butanol) pheophorbide derivative (Ph4-OH) and red light (660 nm) illumination	In human whole blood infected with 3% B. divergens (a bovine-infecting Babesia species), a concentration of 2 µM Ph4-OH coupled to a 10-min illumination, or 4 µM Ph4-OH coupled to a 2-min illumination, reduced parasitemia to undetectable levels.	The first documented evidence of inactivation of a Babesia species by a photochemical treatment in blood. Conditions that effectively eradicate B. divergens lead to 0.1%–0.5% hemolysis.	(156)
INACTINE PEN110 (V. I. Technologies)	In vitro treatment of red blood cells from infected hamsters with 0.01% (vol/vol) PEN110 for 24 hours, or with 0.1% PEN110 for 18 hours, reduced parasitemia to below detectable levels, as determined by blood smear and PCR analyses,and by inoculation of PEN110-treated blood from infected hamsters into naïve hamsters.	− a	(157)
INTERCEPT (amotosalen [S-59]) (Cerus Corp.)	Treatment of B. microti-inoculated human plasma with 150 µM amotosalen and 3 J/cm2 long-wavelength ultraviolet light (UVA; 320- 400 nm) reduced in vivo infectivity by >5.3 ± .4 log.	Coagulation factors and activity were slightly reduced by the treatment but remained within reference ranges. The first proof-of-principle demonstration of the efficacy of INTERCEPT in inactivating B. microti.	(158)
INTERCEPT (amotosalen [S-59]) (Cerus Corp.)	Treatment of human platelet concentrates (resuspended in 65% PAS—35% plasma) containing B. microti-infected mouse blood with 150 µM amotosalen and 3 J/cm2 long-wavelength ultraviolet light (UVA; 320–400 nm) reduced in vivo infectivity by >5.3 log. Likewise, treatment of human plasma containing B. microti-infected mouse blood with 150 µM amotosalen and 3 J/cm2 long-wavelength ultraviolet light (UVA; 320–400 nm) reduced in vivo infectivity by >5.3 ± .4 log.	−	(159)
Mirasol (riboflavin (vitamin B2) and UV light) (Terumo BCT) (formerly Caridian BCT)	Treatment of apheresis plasma and platelets spiked with blood from B. microti-infected hamster by Mirasol technology (riboflavin final concentration ~50 µM; UV light: 285–365 nm, 6.24 J/mL) reduced in vivo infectivity by 4–5 log.	The first proof-of-principle demonstration of the efficacy of Mirasol in inactivating B. microti.	(62)
Mirasol [riboflavin (vitamin B2) and UV light] (Terumo BCT)	Treatment of whole blood spiked with blood from B. microti-infected hamster by Mirasol technology (riboflavin final concentration ~50 µM; UV light: 285–365 nm, 80 J/mL RBC) reduced in vivo infectivity by >5 log.	Notably, a much higher dose of UV is required to inactivate B. microti in whole blood compared to plasma or platelet concentrates. This UV dose required an illumination time of ~40 min, instead of ~10 min for plasma and platelet concentrates.	(160)

^a –, no further remarks.

Deciding when to change interventions

In Babesia non-endemic areas, an important question is when to change TTB risk mitigation strategies. Assessment of transfusion-associated risk is undertaken for novel and emerging pathogens, with a view to inform the need for intervention (161). Blood operators often use a risk-based decision-making (RBDM) process to help determine how and when to change interventions to ensure blood safety (162). The process of RBDM evaluates a series of key characteristics of the pathogen of interest, the available data (Table S1) or drawing on related examples when the data are lacking (Table 6). For example, in Canada, a base scenario estimated one Babesia transfusion transmission every 12.5 years (Table 6) (163). In an RBDM analysis, the key characteristics include (i) the development of significant morbidity or mortality if acquired, (ii) the prevalence in the general and blood donor populations (which are typically different), (iii) the immunopathogenesis of infection (specifically whether there is an appreciable asymptomatic phase during which an infected individual might donate), (iv) the ability of the pathogen to withstand blood processing (e.g., leukoreduction) and storage (e.g., refrigeration), (v) transmissibility via blood transfusion, and (vi) the probability of developing signs and symptoms of the associated infection if transmitted. Should the data support mitigation, additional considerations are the optimal approach of (i) donor selection, (ii) laboratory-based screening, or (iii) pathogen reduction, which, in turn, may be decided in part on the cost-utility of intervention. In Canada, after a risk analysis (163) and then an RBDM, the decision was to not implement routine donor Babesia screening with NAT but to continue Babesia surveillance. Despite this RBDM framework, there are cultural and geographic nuances that account for the variability in risk tolerance and better explain why decisions may be quite different across organizations or countries.

TABLE 6.

Publications describing mathematical models to estimate the risk of TTB

Analytic design	Data sources	Key findings	Reference
Decision tree analysis. Intended to estimate the cost-effectiveness of screening strategies in endemic US states.	Weighted average of published blood donor studies in endemic states. Published estimates of the proportion of donors with Babesia who would have window period infections. Likelihood of developing symptomatic babesiosis after transfusion transmission from donor data.	Predicted 3.6 cases of TTB per 100,000 RBC transfusions with no screening. Universal antibody screening would decrease the rate to less than 0.3 cases/100,000, and with PCR testing to 0.1.	(164)
Decision tree analysis—deterministic. Intended to estimate cost-effectiveness of screening strategies in endemic US states.	Prevalence was estimated from the literature for different test profiles. Transmission probabilities were obtained from the literature. Patient risk proportions were obtained from the literature.	Predicted 32.7 transmissions and 12.9 complicated (scenario 1, considers donor risk only) or 42.4 and 18.4 scenario 2 (considers donor risk and patient risk) in endemic US states. These would be averted by PCR and antibody testing.	(165)
Markov model. Intended to estimate the cost-effectiveness of screening strategies in endemic US states but considers all states in mainland US.	Prevalence by the state was estimated using donor and gen pop prevalence where available, babesiosis medical insurance claims (all US states), and public health-reported cases (all US states). Published donor data on test-positive profiles (antibody and PCR) and test sensitivity and specificity by test-positive profile. Weighted estimate of transmission probability from published studies.	Without testing the four highest prevalence US states would have 20 transmissions/100,000 three unit transfusions, 14.4 per 100,000 would develop mild illness, 0.6 babesiosis requiring hospitalization, and only 0.2 would die. The most cost-effective screening strategies were antibody testing alone,or PCR alone in high-risk US states.	(166)
Monte Carlo simulation. Intended to estimate the risk of a potentially infectious unit being released into inventory in Canada.	Prevalence by region was estimated using public health-reported Lyme disease cases, the ratio of babesiosis vs Lyme disease cases from US public health data, and an adjustment factor based on expert opinion. NAT-positive proportions were obtained from US donor literature. The proportion of NAT positives that are potentially infectious obtained from the literature. A scenario using donor study data was also used.	In the base scenario 0.5, NAT-positive donations would be expected per year (1 transmission every 12.5 years) if incidence were more localized 0.21 NAT positives per year (1 transmission every 25 years). Using donor study data, 4.6 donations would be expected per year (0.81 transmissions per year). Note: the study data were limited by sample size.	(163)

Cost analysis studies in the United States that evaluated outcomes, such as cost-effectiveness and quality-adjusted life years, varied in study design, geographic regions modeled, and types of donor screening technologies. No consensus was found between studies on the cost-effective of regional or nationwide donor screening approaches. However, some form of testing in high-prevalence areas may be beneficial (164 – 166).

Decisions to undertake surveillance may face barriers even as climate change promotes changes in the epidemiology of Babesia (12). Factors that may impede surveillance efforts include the barriers surrounding the collection, storage, and processing of suitable specimens (e.g., whole blood), access to blood donor and clinical laboratory testing, and a lack of resources to support epidemiological studies. These limitations can be overcome through international collaborations that leverage existing expertise and resources and empower local blood operators to make decisions based on current data (90, 92).

Summary of the current state of TTB

Babesia is a tick-borne parasite that upon infection in humans localizes to red blood cells (39). Ticks that transmit human infection include Ixodes spp. but may also include other tick genera depending on the local ecosystem (18, 25, 39, 44 – 46). Humans are dead-end hosts for tick-borne infection, but human-human infection may occur via the transfusion of Babesia-infected red cell products, including cellular products containing residual red cells. Babesia survives within stored blood bags for up to 35 days, and the number of viable parasites required to generate an infection is thought to be extremely low (64). Transfusion recipients are disproportionately at risk of severe or even fatal babesiosis, given their predisposing comorbid risk factors that include extremes of age, cardiorespiratory disease, asplenia, cancer, immunosuppressive therapy, and other immunocompromising conditions. The fatality rate of TTB is more than twice that following tick-borne acquisition (8, 70, 74, 76 – 78). Nearly all reports of blood donor studies originate from the northeastern United States where B. microti is endemic (80 – 82). Currently, there are insufficient data to draw meaningful conclusions regarding the risk of TTB outside of the United States and Canada.

In 2019, the US FDA provided guidance on mandatory TTB risk reduction approaches in at-risk jurisdictions. In those jurisdictions, blood operators must test each donation using a licensed Babesia assay or implement an FDA-approved pathogen reduction technology (13). As of 2023, there is no FDA or Health Canada-approved pathogen reduction device for whole blood or red blood cells. In the United States, the laboratory-based donor screening approach appears successful as donor screening with Babesia NAT has been associated with a significant reduction (although not an elimination) of TTB cases (133, 167). In Babesia non-endemic areas, within the United States as well as globally, blood operators may use an RBDM process to assess how and when to implement or change interventions to ensure blood safety (163, 168).

SUPPLEMENTAL MATERIAL

The following material is available online at https://doi.org/10.1128/jcm.01268-22.

Supplemental Material. jcm.01268-22-s0001.docx.

SUPPLEMENTAL TABLE 1 Blood donor studies and reports of Babesia infection and References

ASM does not own the copyrights to Supplemental Material that may be linked to, or accessed through, an article. The authors have granted ASM a non-exclusive, world-wide license to publish the Supplemental Material files. Please contact the corresponding author directly for reuse.