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Coinfections Acquired from Ixodes Ticks

Epidemic Intelligence Service Program, Office of Workforce and Career Development, Centers for Disease Control and Prevention, Atlanta, Georgia,¹ Acute Disease Investigation and Control, Minnesota Department of Health, St. Paul, Minnesota,² Marshfield Clinic Research Foundation, Marshfield, Wisconsin³

SUMMARY

INTRODUCTION

BIOLOGY AND ECOLOGY OF IXODES TICKS

COINFECTIONS AMONG IXODES TICKS AND MAMMALIAN HOSTS

Prevalence of Coinfecting Pathogens among Ixodes Ticks

North America.

Europe.

Asia and the remainder of the world.

Prevalence of Coinfecting Pathogens among Nonhuman Mammalian Hosts

Transmission Dynamics of Coinfections among Ticks and Reservoir Hosts

Effects of Strain Diversity

COINFECTIONS AMONG HUMANS

Epidemiology of Coinfections among Humans

Prospective studies. (i) Molecular evidence of coinfection.

(ii) Serologic evidence of coinfection.

Serologic studies. (i) Lyme disease-babesiosis coinfection.

(ii) Lyme disease-HA coinfection.

(iii) HA, babesiosis, and triple coinfection.

Laboratory Diagnosis of Coinfections

Pathogenesis and Immunologic Effects

Clinical Manifestations

Lyme disease and babesiosis.

Lyme disease and HA.

Transfusion-Related Tick-Borne Illness

THERAPY

Treatment of HA and LD

Treatment of Babesiosis

STRATEGIES FOR PREVENTING COINFECTIONS FROM IXODES TICKS

RESEARCH NEEDS

ACKNOWLEDGMENTS

REFERENCES

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INTRODUCTION

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Ticks have been implicated as a source of disease for >100 years. In 1893 Smith and Kilbourne offered the first description of a tick-borne disease, establishing that the cattle tick (Boophilus microplus) transmits the protozoan Babesia bigemina, the causative pathogen of Texas cattle fever (182). This dramatic report became the foundation for subsequent work on vertebrate hosts and arthropod vectors. Later work in 1909 by Ricketts recognized the role of ticks as vectors of human disease, with his description of the wood tick, Dermacentor andersoni, transmitting Rocky Mountain spotted fever (165). The first recognition of disease caused by Ixodes ticks occurred in the early 20th century when a Swedish dermatologist reported that the bite of an Ixodes ricinus tick was associated with a characteristic skin lesion near tick bites, termed erythema chronicum migrans (2). In the 1940s, spirochetes were observed in skin lesions, but only isolated cases of erythema migrans (EM) were reported until 1975, when Steere and colleagues investigated a cluster of children with juvenile rheumatoid arthritis living in Old Lyme, Connecticut (192, 193). They observed that the majority of children had illness onset in the summer or fall, and many recalled an expanding rash before the onset of arthritis. Further epidemiologic investigations strongly implicated Ixodes scapularis as the tick vector for Lyme disease (LD) (191). Not until 7 years after the initial recognition was a spirochete (Borrelia burgdorferi) finally isolated from Ixodes ticks by Burgdorfer and colleagues at the Rocky Mountain Laboratories of the U.S. Public Health Service (31).

Since then, newly recognized pathogens and health hazards associated with Ixodes ticks have increased dramatically. We now realize that B. burgdorferi is a genogroup of multiple closely related spirochetes, which have been described throughout the world. The first documented human case of babesiosis occurred in 1957 (181), but only a few isolated cases were reported before 1977, when five cases of Babesia microti infection were identified among residents of Nantucket Island (167). In 1979 the vector for B. microti was identified as an Ixodes tick, and the white-footed mouse (Peromyscus leucopus) was thereafter identified as being a common reservoir for both B. microti and B. burgdorferi (184, 186). Human infections with other Babesia species have since been reported, including Babesia divergens and the unnamed species WA1, CA1, MO1, and TW1 (82, 150, 160, 177).

Human anaplasmosis (HA; previously known as human granulocytic ehrlichiosis) was first reported among patients from Minnesota and Wisconsin in 1994 (12, 39). The etiologic agent, Anaplasma phagocytophilum (previously known as Ehrlichia equi and E. phagocytophila), was detected in blood samples from 12 patients presenting with fever, headache, and myalgias. Subsequent studies confirmed I. scapularis as the vector (147). HA is now known to occur in regions of North America and Europe inhabited by vector-competent species of Ixodes (24, 25, 49, 170, 201, 213). Certain species of Ixodes ticks in Europe (I. ricinus and I. persulcatus) are also capable of transmitting tick-borne encephalitis (TBE) virus, a flavivirus that can cause fatal brain infection among humans (47, 142, 226).

Not surprisingly, because all of these agents can coexist in Ixodes ticks, coinfections have been reported. However, the epidemiology and natural history of coinfections are not fully understood, and the majority of clinicians have limited experience in recognizing or managing them. The purpose of this review is to summarize relevant findings from the medical literature on the occurrence, natural history, and outcomes of coinfections acquired from Ixodes ticks.

BIOLOGY AND ECOLOGY OF IXODES TICKS

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Approximately 865 species of ticks exist worldwide (95), of which approximately 650 species are classified in the family Ixodidae, characterized by the presence of a dorsal plate (scutum). The genus Ixodes includes approximately 245 species, of which 14 are in the ricinus complex (96). This complex includes four species (I. scapularis, I. pacificus, I. ricinus, and I. persulcatus) that account for the majority of Ixodes-vectored human disease. These species are widely distributed throughout the world (Fig. 1) and serve as primary vectors of LD, HA, and babesiosis. In the northeastern and north central United States, I. scapularis is a competent vector of these diseases, able to acquire, transstadially maintain through tick life stages, and subsequently transmit pathogens to susceptible hosts (55, 156, 183, 206, 207). On the West Coast of the United States, the primary vector is a morphologically similar species, Ixodes pacificus (107, 163, 164). In Europe, including the British Isles, I. ricinus is the primary vector for LD, HA, and probably babesiosis (43, 56, 68, 71, 77, 188, 208), but it is largely replaced by I. persulcatus in Eastern Europe and Asia (6, 37, 203).

View larger version (28K): [in this window] [in a new window]   FIG. 1. Approximate geographic distributions of four medically important Ixodes ricinus complex ticks. (Adapted from reference 28a with permission.)

The distribution and abundance of Ixodes ticks are related to multiple factors, including the presence of suitable wooded or brushy habitat and the abundance of hosts for all life stages of the ticks. The resurgence in white-tailed deer populations during the past 30 years might have allowed I. scapularis to expand its range in much of the eastern United States (80, 186, 220). The distributions of tick-borne pathogens and resulting human infections often depend on local tick feeding habits and the distribution and density of small-mammal species that act as competent pathogen reservoirs. For example, the lack of human LD cases in the southern United States might be partially the result of immature I. scapularis ticks commonly feeding on lizards (144), which are incompetent reservoir species for B. burgdorferi (108, 186); in addition, for unknown reasons, I. scapularis ticks in that region do not commonly bite humans (66). Conversely, northern populations of immature I. scapularis feed on reservoir-competent small mammals (e.g., P. leucopus and eastern chipmunks [Tamias striatus]) as well as humans. Reservoir competence, local tick vector feeding habits, and pathogen strain variations each contribute to differences in the geographic distribution of tick-borne diseases.

The risk for tick-borne disease is also closely linked with the life cycle of the Ixodes tick and with vector competency at each life stage. This life cycle involves four life stages (egg, larva, nymph, and adult) and spans 2 years, with tick activity differing dramatically by season and life stage. For example, larval I. scapularis ticks often have peaks in seasonal activity during early and late summer, whereas the nymph stage is most active from late spring through midsummer (137, 221). Adult I. scapularis ticks are abundant during the early fall and are active again during spring months if they did not feed in the fall. Transmission of LD, HA, and babesiosis usually occurs during the relatively short period of the nymph stage when the tick is active (145). The nymphs' small size (approximately 1 mm) allows them to often feed undetected on humans long enough to transmit these pathogens. Adult ticks are larger and more likely to be detected and removed before disease transmission, whereas host-seeking larvae are uninfected and thus epidemiologically unimportant.

The feeding behavior of Ixodes ticks at each life stage has an impact on the risk for tick-borne infection and coinfection among humans. All Ixodes species of public health importance are three-host ticks that must find a new host at each life stage. During each life stage after hatching (larva, nymph, and adult), an Ixodes tick takes one blood meal, which typically requires 3 to 5 days to complete. Certain Ixodes ticks are host specific, whereas others feed on different host species. Those with nonspecific feeding habits, (e.g., I. scapularis, I. pacificus, I. ricinus, and I. persulcatus) not only feed on species that are reservoirs for multiple tick-borne pathogens (e.g., small mammals) but also will readily bite humans. Therefore, nonspecific feeders might be more important as vectors of human disease than host-specific ticks, which are less likely to bite humans.

When feeding on an infected small-mammal host, tick larvae and nymphs can take up one or more pathogens, which might be transmissible during subsequent blood meals. Larvae are generally not infected with B. burgdorferi, A. phagocytophilum, or B. microti upon hatching; transovarial passage of these pathogens from adult females to eggs has not been consistently demonstrated or is considered insignificant (91, 136, 148, 154, 171, 228). However, transovarial transmission of B. divergens from adult I. ricinus ticks to larvae does occur (57, 207) and is also believed to be important in maintaining the life cycle of other tick-borne viral and rickettsial pathogens (e.g., TBE virus, spotted fever group rickettsia) (32, 162). Following acquisition of either LD, HA, or Babesia, transstadial transmission (i.e., from larva to nymph or from nymph to adult tick) occurs. After molting, nymphs and adult ticks infected in a previous life stage emerge infective and may transmit disease to susceptible hosts during subsequent feedings. Adult female ticks require a blood meal to develop their egg mass and commonly seek a large-mammal host for their third and final blood meal.

COINFECTIONS AMONG IXODES TICKS AND MAMMALIAN HOSTS

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North America. Molecular evidence of coinfection with multiple human pathogens has been demonstrated for Ixodes ticks sampled from select geographic areas of California, Wisconsin, and the northeastern United States (Table 1). The prevalence of dually infected ticks appears highest among I. scapularis ticks from regions of LD endemicity in the northeastern United States, with reported prevalences of 28%. Studies from other North American regions have generally reported lower prevalences of dually infected Ixodes ticks. In Wisconsin, 2% of I. scapularis adult ticks were coinfected with B. burgdorferi and A. phagocytophilum (147). In northern California, approximately 1% of both I. pacificus nymph ticks from deciduous woodlands (109) and I. pacificus adult ticks from coastal regions (86) were dually infected with B. burgdorferi and A. phagocytophilum (Table 1). Fewer studies have attempted to identify simultaneous infection with three tick-borne pathogens, B. burgdorferi, B. microti, and A. phagocytophilum. These studies weakly suggest that molecular evidence from Ixodes ticks of dual infection with B. burgdorferi and A. phagocytophilum appears more common than B. burgdorferi-B. microti or B. microti-A. phagocytophilum coinfections, although geographic differences do exist (179, 188, 206, 213). Triple coinfection appears to be even less common among Ixodes ticks (Table 1). None of the I. scapularis ticks collected in an area of LD endemicity in New Jersey were demonstrated to have triple coinfection with these pathogens, whereas 4% were dually infected (1). Other researchers have not identified molecular evidence of triple coinfection among Ixodes ticks, despite reporting a dual-pathogen prevalence of 1% to 10% (88, 206, 213). Taken together, these few studies indicate that dual infection with any combination of B. burgdorferi, B. microti, and A. phagocytophilum occurs in 1% to 28% of Ixodes ticks from regions of LD endemicity in the United States and in <1% to 13% of sampled European Ixodes ticks (Table 1). Triple coinfection is rarely detected in geographic regions where all three tick-borne diseases are endemic and likely represents an incident occurrence of <1%.

A limited number of studies have attempted to detect molecular evidence of tick coinfection with Babesia species. From northwestern Poland, coexistent DNA of B. burgdorferi sensu lato species and B. microti was identified for 3% of sampled I. ricinus female adult ticks but only 0.1% of nymphs (180). Among questing I. ricinus ticks collected in northern France, 2% had evidence of coinfection with B. burgdorferi sensu lato and Babesia species (77). Of note, a limited number of European studies have attempted PCR detection of all three coinfecting pathogens in I. ricinus ticks; among these studies, only one report, from northwestern Poland, demonstrated a 1% prevalence of all three pathogens—B. burgdorferi sensu lato, B. microti, and A. phagocytophilum—among I. ricinus ticks (179).

Substantially less is known about the prevalence of dual pathogens among I. persulcatus ticks. Among I. persulcatus adult ticks collected from St. Petersburg, Russia (6), 1% had molecular evidence of coinfection with B. burgdorferi sensu lato and either B. microti or A. phagocytophilum. Triple infection was rarely demonstrated; 0.3% of sampled I. persulcatus ticks had evidence by PCR of TBE virus, B. burgdorferi sensu lato, and either B. microti or A. phagocytophilum. None of the I. persulcatus ticks had triple coinfection with B. burgdorferi sensu lato, B. microti, and A. phagocytophilum.

Asia and the remainder of the world. Coinfection of I. persulcatus ticks has been reported from the forest areas of northeastern China (37), where LD is highly endemic (5, 205). Of 1,345 adult and nymph I. persulcatus ticks, 33.8% were infected with B. burgdorferi, 4.6% with A. phagocytophilum, and 0.5% with both pathogens (37). Coexistence of both pathogens had not been previously reported for I. persulcatus ticks from Asia. Korenberg and colleagues reported a 6% prevalence of coinfection with TBE virus and Borrelia species among I. persulcatus Eurasian ticks (99). The prevalences of TBE virus and Borrelia in ticks appeared independent, with no apparent effect on each other (98).

Overall, information is limited or nonexistent on the prevalence of pathogens among Ixodes ticks in Asia, Central and South America, Oceania, and Africa. Furthermore, despite reports of human babesiosis from countries such as China (71), Taiwan (177), Japan (8, 168), Colombia (166), Mexico (71), Egypt (127), and South Africa (35), coinfection of Ixodes ticks with Babesia species and B. burgdorferi or A. phagocytophilum has not been reported outside sampled regions of LD endemicity in Europe and the United States.

Studies have reported the prevalence of coexisting tick-borne pathogens among nonhuman mammalian hosts. Among white-footed mice (P. leucopus) captured in Lyme, Connecticut, 50% had evidence of past or present infection with B. burgdorferi, B. microti, and A. phagocytophilum (187), confirming earlier findings of antibodies to these pathogens among mice from Connecticut (116). B. burgdorferi and A. phagocytophilum DNAs were simultaneously detected among 7% of I. scapularis ticks allowed to feed as nymphs on wild-caught P. leucopus in Connecticut (112). Naturally occurring coinfection with B. burgdorferi and B. microti has also been documented for P. leucopus mice captured in the upper Midwest (85). Among these and perhaps other populations of P. leucopus mice, B. microti infection was strongly associated with concurrent B. burgdorferi infection (85). In areas of the western United States, coinfection with A. phagocytophilum and B. burgdorferi has been demonstrated among additional rodent species, including deer mice (Peromyscus maniculatus), Mexican wood rats (Neotoma mexicana), and prairie voles (Microtus ochrogaster) (224). In Colorado, B. microti DNA has been commonly detected among prairie voles as well (33). Both B. burgdorferi and B. microti are considered to cause long-lived infections among rodent reservoir hosts (153, 185), but less is known about the duration of A. phagocytophilum infections among reservoir hosts.

In Europe, additional studies have demonstrated the presence of Francisella tularensis as a coinfecting pathogen among reservoir animals. Christova and Gladnishka evaluated captured urban rodents (e.g., Rattus rattus, Mus musculus, and Apodemus agrarius) for infection with F. tularensis, B. burgdorferi sensu lato, and A. phagocytophilum (40). PCR assays yielded evidence of F. tularensis in 22% of captured rodents, whereas B. burgdorferi and A. phagocytophilum DNAs were detected in specimens from 26% and 8% of rodents, respectively. Overall, the prevalence of coinfection with F. tularensis and either B. burgdorferi or A. phagocytophilum was 7%. A similar study of small terrestrial mammals captured from a region of the Austrian and Slovakian borderland where LD and TBE are endemic revealed a coinfection prevalence of 0.5% with B. burgdorferi sensu lato and F. tularensis (214). Taken together, evidence of coinfection among rodent hosts has increased, yet information on the prevalence, intensity, or duration of dual and triple infections among these and other reservoir hosts remains limited.

Transmission cycles among ticks and vertebrate hosts are perpetuated when ticks transfer pathogens between susceptible hosts (horizontal transmission) but cannot be sustained when transmission is directed toward dead-end hosts incapable of experiencing high levels of the organism in blood (tangential transmission). Reservoir host responses to infection with a tick-borne pathogen differ, depending on the specific agent and host, and this interaction has a direct impact on transmission dynamics. For example, parasites of red blood cells (e.g., Babesia spp.) are often associated with long-term, relatively asymptomatic infection of the reservoir host. These chronically infected animals can provide numerous opportunities for feeding ticks to acquire infection. In contrast, viral and bacterial infections often either are fatal or induce an immune response in the reservoir host that limits the time during which the pathogen is circulating in high numbers in the peripheral blood. In those situations where fewer opportunities exist for feeding ticks to acquire infection, the tick becomes the crucial link in maintaining the enzootic cycle in nature, by passing organisms either between different stages of tick development (transstadial maintenance from larva to nymph or from nymph to adult), between generations (transovarial transmission from an adult female to her eggs), or from one tick to another during cofeeding in close proximity on the same host (149).

Theoretically, coinfection with Ixodes-associated pathogens has the potential to modulate transmission dynamics at multiple points in the transmission chain. These include alterations in the efficiency of transmission from rodent to tick or from tick to vertebrate, cooperative or competitive pathogen interactions, and increasing or decreasing disease severity among hosts (210). Several laboratory studies have been used to quantify these potential interactions, and the results have been conflicting.

For example, Levin and Fish investigated whether previous infection of ticks with either Borrelia or Anaplasma affects the acquisition and transmission of a second pathogen. They fed Anaplasma-infected I. scapularis nymphs on Borrelia-infected mice (and vice versa) and measured the efficiency of previously infected nymphal ticks at acquiring a second pathogen and transmitting one or both agents to susceptible hosts. No evidence of interaction between the agents of LD and human anaplasmosis among I. scapularis ticks was found with regard to acquiring or transmitting these infections (113). A murine model of coinfection, however, reveals that dual infection with B. burgdorferi and A. phagocytophilum alters immune responses and increases the pathogen burden, such that an increased bacterial burden resulted in increased pathogen transmission to the vector (87, 209).

Antigenic variation in major surface proteins of tick-borne bacterial pathogens is one of the most important mechanisms for evasion of the host immune response and can result in persistent infection. This can be accomplished by different mechanisms. For example, borreliae generate antigenic diversity of specific coat proteins (vmp/vls) through a process of recombination termed gene conversion (16, 227). Gene conversion is usually widespread among tick-borne bacterial pathogens and allows organisms to retain a complete set of variable antigen genes. In selected instances, gene conversion is complete, and all epitopes of an antigen are replaced. On other occasions, partial replacement occurs at hypervariable regions of proteins.

Antigenic variation can also occur at the level of gene transcripts. Gene expression of a variable antigen can be activated at one locus and inactivated at another. This is a reversible process that does not involve changes in DNA at the loci themselves. Conversely, certain DNA rearrangements involve recombination between short direct repeats common to two or more alleles and result in the loss of an allele in the process. Finally, antigenic variation can be generated by accumulation of point mutations among multiple genes. These mutations, along with recombination or reassortment between two different strains infecting the same host, are essential for generating genetic variation among select tick-borne pathogens.

In animal models, B. burgdorferi strain variation has been demonstrated to alter the risk of disease transmission. Derdakova and colleagues investigated the interaction between two strains of B. burgdorferi in a laboratory system of P. leucopus mice and I. scapularis ticks. Two groups of mice were infected with either strain BL206 or strain B348 of B. burgdorferi. Two weeks later, experimental mice were challenged with the opposite strain. Transmission of both strains was assessed by xenodiagnosis with uninfected larval ticks at weekly intervals. Fewer dual infections were observed among xenodiagnostic ticks, and BL206 was transmitted more efficiently than B348. These findings suggest that certain B. burgdorferi strains (e.g., BL206) might be preferentially maintained in transmission cycles between Peromyscus mice and ticks, whereas other strains are maintained in alternate tick-vertebrate host transmission cycles (53). However, whether strain variation in B. burgdorferi affects the transmission dynamics of other tick-borne pathogens is unclear.

Strain variation has critical implications for preventing tick-borne infections, including vaccine development and serologic tests. If variable antigens are the intended targets for immune prophylaxis, then certain vaccines for pathogens transmitted by Ixodes ticks will need to be multivalent. B. burgdorferi strain and genospecies diversity is a more acute issue in Europe than in North America and therefore presents greater challenges for vaccine development. Which epitopes to include or exclude in vaccines might not be obvious; too few antigens might provide insufficient protection, while too many epitopes might render development of an effective vaccine impractical. Furthermore, when different geographic areas require different vaccine formulations, the market might not be sufficiently large to support product development. Similar concerns surround the laboratory diagnosis of tick-borne infections, especially with regard to immunoserologic testing; determining the best combinations of epitopes to include in an enzyme-linked immunosorbent assay (ELISA) or similar assay for optimal sensitivity and specificity is difficult (161).

COINFECTIONS AMONG HUMANS

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Human coinfection with tick-borne pathogens can occur after attachment of a single tick infected with multiple pathogens or from concurrent single-pathogen tick attachments. Both of these scenarios potentially can result in human coinfection and might not be easily differentiated from sequential infection by pathogens occurring at different points in time. Individual differences in innate and acquired immunity, as well as differences in personal behaviors, occupation, activities, and place of residence, contribute to one's risk for acquiring tick-borne infections. Studies have reported that age-related differences exist among patients with diagnosed babesiosis alone (104), those with HA alone (18), and those at risk for coinfection with LD and HA (3). However, at least one prospective study of tick-borne coinfections demonstrated no substantial differences by age or sex (104).

Epidemiologic knowledge is further limited in Europe and North America by the common use of seroprevalence data, with little ability to differentiate sequential or past infections from simultaneous infections. Additional limitations of seroprevalence studies exist (e.g., inappropriate cutoff values, false-positive and false-negative reactions, and possible cross-reactivity between tick-borne pathogens such as A. phagocytophilum and B. burgdorferi) which should be considered in interpreting the epidemiologic conclusions of these studies. In contrast, epidemiologic studies that use prospective seroincidence data or molecular methods of DNA detection provide a more accurate picture of the incidence of coinfections; these studies, however, are less common. Taken together, epidemiologic studies demonstrate that the majority of coinfections acquired from Ixodes ticks in North America and Europe include infection with B. burgdorferi, for reasons that need further investigation.

Prospective studies. (i) Molecular evidence of coinfection. In prospective studies, the incidence of coinfection appears highest among persons with LD; 4% to 45% of LD patients from regions where LD is endemic are coinfected with either HA or babesiosis. In a 1997-to-2000 New England study, patients who presented during the summer months with an EM rash or influenza-like illness were prospectively enrolled; they submitted blood samples for tick-borne, pathogen-specific serologic and PCR assays (104). One hundred ninety-two (62%) of 310 patients in this study had at least one tick-borne disease; 75 (39%) of these 192 patients had coinfections. LD and babesiosis accounted for the majority (81%) of tick-borne coinfection scenarios, followed by LD-HA coinfection (9%), triple coinfection (LD, HA, and babesiosis [5%]), and lastly babesiosis-HA coinfection (4%). In this particular study, 161 patients had diagnoses of acute LD; 45% of these LD patients demonstrated simultaneous evidence of coinfection with B. microti or A. phagocytophilum.

Other prospective studies have reported lower rates of acute coinfection. Approximately 10% of 240 LD patients from southern New England had either PCR, serologic, or direct microscopic evidence of coinfection with B. microti (106). In a 4-year prospective study in Rhode Island and Connecticut, 2 (2%) of 93 patients with a culture-proven Borrelia burgdorferi EM skin lesion had PCR or immunoglobulin G (IgG) seroconversion evidence of coinfection with B. microti, and 2 (2%) had evidence of coinfection with A. phagocytophilum (194). A prospective Wisconsin study of patients with EM indicated a higher prevalence of coinfection with A. phagocytophilum, with 11 (12%) of 94 patients with EM demonstrating laboratory evidence (serologic or molecular) of dual infections (20). Notably, approximately 20% of patients with LD do not develop a rash (195, 200), and these persons were not included in either prospective study.

(ii) Serologic evidence of coinfection. In the only prospective seroincidence study performed to date, 671 persons with high-risk exposures in a region of New York where Lyme borreliosis is endemic participated in a 1-year study (84). Nineteen persons (2.8%) seroconverted to A. phagocytophilum, B. burgdorferi, B. microti, or Rickettsia rickettsii. However, incident cases of coinfection were not observed, because no participants seroconverted to dual pathogens during the 1-year follow-up. Five participants (0.7%) had evidence of prior exposure to dual pathogens on their baseline sera. This study suggested that the absolute risk for dual infections is low, even among populations at high risk. Although the absolute risk for coinfection appears to be low, this risk differs by geographic region and by level of human and tick activity. Not surprisingly, when coinfection is reported, it is from regions of Lyme borreliosis endemicity, and coinfection occurs most commonly among patients with LD. This indicates that patients with one documented tick-transmitted infection might be at increased risk for infection with another pathogen. At present, coinfection with A. phagocytophilum and B. microti and triple coinfections are rarely reported, even in prospective studies.

Serologic studies. (i) Lyme disease-babesiosis coinfection. Geographic areas where LD and babesiosis are endemic, particularly regions of New England and the mid-Atlantic states, have long been associated with reported serologic evidence of both B. burgdorferi and B. microti among humans. Serologic confirmation of concurrent babesiosis and LD was first reported in 1983 for an asplenic male aged 36 years, from Shelter Island, N.Y., who experienced recurrent fevers, erythema chronicum migrans, and monoarticular arthritis (72). Within 2 years, additional reports confirmed the simultaneous occurrence of Lyme borreliosis and babesiosis (119, 198). In a retrospective study of persons residing in areas of LD endemicity in New York and Massachusetts during 1978 to 1984, approximately 50% of patients with confirmed babesiosis had antibodies to B. burgdorferi (22). In the same study, 66% of patients who fulfilled clinical and serologic criteria for LD had IgM and IgG antibodies to B. microti (22). Additional studies have reached similar conclusions, namely, that the seroprevalence of B. microti is highest among persons with prior or active LD (105, 118, 217). For instance, on Nantucket Island, the estimated population seroprevalence of both B. burgdorferi and B. microti is 3.5%; however, 26% of Nantucket Island residents who were seropositive for LD also had serologic evidence of prior B. microti infection (217).

Other studies from regions of Babesia and LD endemicity in the northeast and mid-Atlantic United States have also demonstrated serologic evidence of B. microti infection among persons with LD, although generally in the 2%-to-12% range (Table 2). Febrile Connecticut residents with hematologic abnormalities and exposure to tick-infested areas were evaluated for antibodies to tick-borne pathogens (118). Twenty-two of 180 (12.2%) seropositive persons had dual antibodies to B. microti and B. burgdorferi, and 15 (8.3%) had antibodies to E. equi (A. phagocytophilum) and B. burgdorferi. In Wisconsin and Minnesota, 2 (2%) of 96 patients with laboratory-confirmed LD demonstrated immunoserologic evidence of B. microti infection (128). On the West Coast, the recently identified Babesia species WA-1 was determined in one study to be a coinfecting pathogen; 60 (23.5%) of 255 LD patients tested positive for antibodies to the WA-1 piroplasm (199).

(ii) Lyme disease-HA coinfection. Serosurveys indicate that simultaneous occurrence of antibodies to B. burgdorferi and A. phagocytophilum is relatively common. In Wisconsin, Minnesota (20, 128), and regions of the northeastern United States (3, 50, 84), seropositivity for both pathogens ranged from <3% to 26% (Table 2). In a serosurvey of residents of Connecticut and Rhode Island performed by an ELISA and Western blotting for B. burgdorferi and an ELISA (with a recombinant HGE-44 protein) for A. phagocytophilum, 2 (4%) of 52 patients had a positive IgG response to each, and 7 (21%) of 34 patients with a positive IgM response to B. burgdorferi also had a positive IgM response to A. phagocytophilum (50). In a study from Westchester County, New York, Aguero-Rosenfeld and colleagues demonstrated that 45 (26%) of 175 B. burgdorferi-seropositive subjects had antibodies to A. phagocytophilum (3). The same study found that 9 (21%) of 42 patients with culture-confirmed Lyme borreliosis were seropositive for A. phagocytophilum. It should be noted, however, that this study also demonstrated a 5%-to-11% background rate of seropositivity for A. phagocytophilum among healthy B. burgdorferi-negative children and adults, suggesting potential limitations of serologic testing (e.g., false-positive reactivity, low cutoff values) (3). False-positive IgM responses to B. burgdorferi are now recognized to occur also in response to A. phagocytophilum infection (222), such that determining B. burgdorferi-A. phagocytophilum coinfection from serology alone is problematic.

In Europe, human HA infection was first reported for a Slovenian woman, aged 70 years, with evidence of potential coinfection with B. burgdorferi sensu lato determined through a rise in the IgG antibody titer (151). Serologic evidence of HA infection has since been reported widely across Europe, in more than 17 countries. Seroprevalence rates among examined populations range from zero or low to 28% (201); however, nonstandardized serologic tests for A. phagocytophilum and different diagnostic approaches make it difficult to fully interpret and compare these different European studies. The highest number of incident cases of HA has been reported in Central Europe (Slovenia) and Sweden, and seroepidemiologic evidence of HA infection has been reported to be higher among persons frequently exposed to ticks (e.g., forestry workers) and among patients with Lyme borreliosis or TBE. Despite this, well-documented, clinically compatible cases of HA have rarely been reported from Europe, and A. phagocytophilum has yet to be isolated from European patients. Potentially infected persons identified by serologic testing often appear to have had asymptomatic infections (48, 67, 146, 158). These factors have led to speculation that European HA might represent a milder illness, possibly related to strain variants, or that serologic testing might be detecting cross-reacting pathogens rather than A. phagocytophilum (25, 70).

Evidence of potential coinfection with the pathogens of LD and HA has since been demonstrated in Belgium (73), the Czech Republic (92), Germany (115), Italy (169), Norway (13), Poland (81), Slovenia (10), Switzerland (28), Sweden (24), and the United Kingdom (202) (Table 2). Studies indicate a range of coinfection prevalences, from 3.2% among permanent residents of the Koster Islands in Sweden to 17% among LD-seropositive individuals in Switzerland (28, 61). A serosurvey of 1,515 persons representing different risk categories for tick exposure in Switzerland indicated that the highest HA seroprevalence occurred among persons who were seropositive for B. burgdorferi (13%) or central European TBE virus (20%) (159).

(iii) HA, babesiosis, and triple coinfection. Only a limited number of studies have attempted to document either dual infection with HA and B. microti or triple coinfection with these two agents and LD. Among 192 patients with confirmed tick-borne illness from Nantucket, Rhode Island, and Connecticut (104) during the months of May through September, 1997 to 2000, dual infection with HA and babesiosis was detected for three (1.6%) persons and triple coinfection for four (2%) persons. In a different study by Magnarelli and colleagues, dual infections with HA and B. microti (n = 1) and triple coinfections (n = 2) were noted for <1% of 375 febrile patients in Connecticut suspected of having a tick-borne illness (118). Within the United States, the highest prevalence of HA-babesiosis dual infections has been reported in Wisconsin, where 7% of patients with confirmed or probable A. phagocytophilum infection demonstrated a fourfold change in antibody titers to B. microti on paired sera samples (19). Overall, evidence of triple coinfection is rare, with the majority of studies reporting no patients to 2% of patients with a tick-associated illness demonstrating laboratory evidence of infection with B. microti, A. phagocytophilum, and B. burgdorferi combined (Table 2).

A common theme is that patients with LD, HA, babesiosis, TBE virus, and other tick-transmitted diseases can all present with relatively nonspecific influenza-like illnesses. In these instances, the choice of laboratory tests should be guided by a thorough patient history and physical examination that documents evidence of tick exposure, place of residence, recent travel, and objective signs and symptoms of tick-borne infection. The decision whether or not to pursue specific laboratory testing can then be based on knowledge of which tick-borne diseases are endemic in a particular area and, more importantly, can be made after thoughtful assessment of the probability that the patient actually has one or more tick-transmitted infections. For example, according to the guidelines of the American College of Physicians, patients with vague subjective complaints (e.g., headache, fatigue, and myalgia) are considered to have a low pretest probability for LD and should not undergo antibody screening by ELISA or immunofluorescence assay (IFA), because the majority of positive results will be false positives as a result of cross-reactivity with other microorganisms or disease conditions (211). This is a critical concept, because an exaggerated perception of risk by patients and health care providers can result in substantial amounts of unnecessary testing and associated expense.

Despite those limitations, after it is established that a patient is at moderate to high risk for having one or more tick-transmitted infections, laboratory testing is indicated with only limited exceptions. Patients presenting with typical primary or secondary EM in areas where LD is endemic can often be treated empirically, and the diagnosis does not require laboratory confirmation. Additionally, when these patients are treated with an antimicrobial agent (e.g., doxycycline), performing laboratory testing to document coinfection with HA is neither cost-effective nor necessary, because therapy is highly effective for both agents. In the majority of other clinical situations, laboratory testing is required for definitive diagnosis and to guide therapy.

Among the most useful laboratory tests for tick-borne infections are complete blood counts and peripheral blood smears along with tests of liver function. For patients with HA, leukopenia, lymphopenia, granulocytopenia, and elevated liver enzymes are commonly observed. Anemia is common in babesiosis, and thrombocytopenia is frequently evident in babesiosis and HA infections. Babesiosis and HA can often be diagnosed directly by observing organisms on Giemsa-stained smears of peripheral blood. For patients with intact spleens, 1% to 10% of erythrocytes might show B. microti ring forms on thin blood smears; this proportion may be as high as 80% for asplenic patients (89, 126). The laboratory should screen multiple slides before considering a smear to be negative. Manual microscopy is a subjective process, and the accuracy of the examination depends on the vigilance and experience of the observer, the intensity of parasitemia, and the timing of evaluation relative to illness onset. Intracellular babesia might be confused with Howell-Jolly bodies (121); conversely, false-positive results can occur when inexperienced observers mistake platelets or staining artifacts for piroplasms within erythrocytes or anaplasmal morulae within granulocytes. A minority of HA patients with early, mild infection had intragranulocytic morulae detected on smears (21), in contrast with symptomatic, untreated patients whose smear results were evaluated after several days of fever (12). With development of more sensitive molecularly based techniques for diagnosing B. microti and A. phagocytophilum, PCR is increasingly relied on to detect infection among patients with low pathogen loads and negative blood smears.

For the majority of tick-borne infections, laboratory diagnosis is often made by detection of IgM antibodies in specimens obtained during acute illness or by observing an increase in IgG antibody titers between acute- and convalescent-phase samples taken 10 to 14 days apart. A significant advantage of this approach is that immunoserologic testing is widely available and is usually cost-effective. When seeking laboratory confirmation of a tick-borne disease, health care professionals should utilize a licensed laboratory that employs strict quality control and is experienced in antibody testing. Multiple testing formats are available, including ELISA, IFA, and immunoblotting. For such diseases as LD, the interpretation of immunoserologic testing has become standardized (29).

In the United States, suspected cases of extracutaneous LD are often evaluated by the CDC's two-step protocol, where positive or equivocal screening results by ELISA or IFA are confirmed by a standardized immunoblot. This approach improves specificity and provides sufficient information to allow rational patient management decisions in the majority of cases. In contrast, for Europe and Asia, development of a uniform approach for the immunoserologic evaluation of LD is complicated, because organisms from three species of the B. burgdorferi sensu lato genogroup can cause infections. Within these species, substantial antigenic variation exists (75). For the best performance, immunoserologic assays need to be developed for defined geographic areas on the basis of specific species and strains of B. burgdorferi sensu lato genogroup organisms that are endemic to each area.

This review does not discuss the performance characteristics of each of the immunoserologic assays available for Ixodes-associated infections, but a number of concerns related to this type of testing warrant emphasis. Clinicians should recognize that performance characteristics for these tests differ, depending on the type and quality of the antigens incorporated into each test. Cutoff values for positive results differ among laboratories; furthermore, the patient population that is being tested and the prevalence of specific infections in a particular geographic area will affect the sensitivity and specificity of the test. Laboratories should provide physicians with detailed information on the performance characteristics for each of the assays they provide.

Culture isolation of Ixodes-associated pathogens from clinical specimens provides direct evidence of infection but can be time-consuming and expensive and is usually limited to special circumstances. Examples of this include confirmation of infection caused by pathogens in areas where they have not previously been endemic and diagnosis of reinfection for patients for whom the results of immunoserologic testing might not be interpretable. The techniques involved often require special culture media, cell lines, animal inoculation, and a high level of biocontainment that is not practical for the majority of clinical laboratories. Sample requirements can also be stringent. Clinicians should contact their reference laboratory for guidance if isolation of these pathogens is being considered.

Laboratories have often turned to molecular assays in an attempt to increase sensitivity and specificity and to decrease the turnaround time for laboratory results. PCR assays are available for detecting nucleic acids of the agents for LD, HA, babesiosis, and TBE. An advantage of molecular detection is the ability to diagnose early infections before the appearance of serum antibodies, without the delay associated with culture isolation. However, these assays have limitations as well, and assessing the probability of a tick-borne infection for PCR-based tests is as important as assessment for immunoserologic assays. False-positive and false-negative results can occur for different reasons, and results need to be interpreted in the context of the clinical situation. Transient or limited numbers of tick-borne organisms (e.g., HA or babesiosis) within the sampled material might yield a false-negative test result. Providers should be aware that there has been little standardization of molecular assays for tick-borne infections across laboratories, and performance characteristics are highly variable. Therefore, in the majority of cases, a negative PCR or other molecular test result does not exclude the possibility that an infection is present. One circumstance where PCR has been evaluated extensively and is especially useful is the case of LD-associated arthritis. Determining whether chronic arthritis is caused by persistent infection or a prolonged immunologic response is difficult. A negative PCR result from joint fluid in this instance supports an immune-mediated etiology for persistent arthritis rather than an active infection requiring additional antimicrobial therapy.

Coinfection with B. burgdorferi and B. microti has also been demonstrated to have immunologic effects in animal models, including alteration of the Th1 cell response and increased severity of arthritis (129). In another mouse model, dual infection with B. burgdorferi and B. microti appeared to follow independent courses (45). When young immunocompetent, young asplenic, young BALB/c, and aged C3H/HeN mice were coinfected with B. burgdorferi and B. microti, babesiosis followed its normal course of infection without evidence of increased severity, as determined by the percentage of parasitemia and other clinical and laboratory parameters. LD also followed its usual course and severity among coinfected mice compared with singly infected control subjects, with no increase in spirochete dissemination or arthritis. In summary, the immunologic and pathological effects in animal models are often not generalizable to humans, and further investigation is needed to determine the clinical implications of these findings.

Persons coinfected with B. burgdorferi and B. microti do not appear to be at greater risk for spirochete dissemination or LD complications, despite the fact that B. burgdorferi DNA persists longer in the blood of coinfected patients after treatment (106). At least one prospective study demonstrated no evidence of increased joint, cardiac, or neurologic disease or hospitalization among coinfected patients compared with patients with LD alone (104). This study's findings are consistent with a similar lack of increase in musculoskeletal and neurologic effects noted in other published reports of coinfected patients (106, 217). Case reports have discussed a more dramatic, severe clinical course among coinfected patients, but these likely reflect a bias toward reporting cases with more severe or unusual manifestations (119, 131, 143, 204). All studies to date have been limited in their power to detect potentially subtle differences in outcomes because of their relatively small numbers of patients. Whether clinical outcomes differ among partially treated or untreated patients with coinfection compared with those with LD alone remains unanswered.

In summary, possible dual infections with B. burgdorferi and B. microti should be considered for patients with a diagnosed tick-borne illness whose exposure occurred in areas where both diseases are endemic. Hematologic findings, such as anemia and thrombocytopenia, are uncommon among patients with LD alone and can be indicators of Babesia coinfection (20, 76, 104). Tick-borne diseases rarely occur during influenza season. Thus, the clinician should consider coinfection for any LD patient complaining of marked influenza-like symptoms, or if the patient demonstrates unexplained splenomegaly, anemia, thrombocytopenia, or a failure to respond to antimicrobial therapy directed against B. burgdorferi.

Lyme disease and HA. A limited number of case reports describe B. burgdorferi-A. phagocytophilum coinfections that have been confirmed by molecular methods or visualization of intragranulocytic morulae. Serologic evidence alone is often insufficient for confirming dual infection, because patients with acute HA infection might have a false-positive LD serology (222). The presence of IgM or IgG antibodies to B. burgdorferi might also represent prior asymptomatic infection. In an LD vaccine trial in the United States, 30 (11%) of the 269 study participants who acquired LD had asymptomatic IgG seroconversion to B. burgdorferi (196). Several seroprevalence studies in Europe have demonstrated that >50% of B. burgdorferi-seropositive persons do not recall having any symptoms of LD (64, 74, 139). Furthermore, levels of IgM and IgG antibodies to B. burgdorferi decline slowly after treatment, and these antibodies have been demonstrated to persist for months to years after clinical cure (65, 94). Thus, LD infection might be incorrectly diagnosed on the basis of a positive serologic test and should be considered significant only if accompanied by a characteristic clinical picture.

A small cohort of 7 Rhode Island patients coinfected with B. burgdorferi and A. phagocytophilum reported significantly more chills, sweats, headaches, arthralgias, and sore throats than 89 patients with LD alone (104). Coinfected patients reported a significantly higher mean number of symptoms and a longer duration of symptoms. Although coinfection with B. burgdorferi and A. phagocytophilum might result in more severe or persistent symptoms (131, 147), no evidence exists of increased dissemination of the LD spirochete among these coinfected patients (104). One-third of patients with HA might have a cough (12), which is rarely reported in cases of LD (133). Leukopenia and thrombocytopenia are commonly reported in HA (12, 104) but would be unusual for LD patients (133, 135, 218). Elevated liver transaminase levels can occur in both Lyme borreliosis and HA (12, 133).

As with babesiosis, clinicians should consider additional laboratory tests for HA when LD patients demonstrate a more intense and persistent array of nonspecific, influenza-like symptoms, especially fever, chills, and headache. Coinfection with HA is also suggested when LD patients fail to respond to appropriate ß-lactam antimicrobial therapy (amoxicillin, ampicillin, or ceftriaxone) (104, 131) or demonstrate laboratory evidence of neutropenia and thrombocytopenia (14, 104). For suspected coinfection with HA or babesiosis, clinicians might consider a complete blood count with a Giemsa-stained blood smear a reasonable initial evaluation.

Nonetheless, viable B. burgdorferi has been identified in refrigerated packed red blood cells, whole blood, and frozen plasma stored for >1 month (11, 63, 134), and public health concern exists regarding the possible risk for transfusion-related transmission of LD. Less documentation exists on the viability of A. phagocytophilum in stored blood products; at least one study documented that presumptive A. phagocytophilum remained viable in refrigerated blood for 18 days (93). At present, there are no known case reports on dual tick-borne infections transmitted through transfusion of blood products.

THERAPY

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Under special non-life-threatening circumstances, rifampin can be considered an alternative to doxycycline for treatment of HA (70). Both rifampin and levofloxacin demonstrate excellent in vitro activity against A. phagocytophilum (90); however, clinical experience and documentation of effectiveness are limited for rifampin and nonexistent for levofloxacin. The use of rifampin in treating HA during pregnancy (30) and for two pediatric patients (102) has been described; no data exist regarding the effectiveness of levofloxacin in treating HA. Chloramphenicol demonstrates suboptimal in vitro activity against A. phagocytophilum (122), and treatment failures with chloramphenicol have been reported (60). A. phagocytophilum is resistant to numerous antimicrobials commonly used in clinical settings, including aminoglycosides (gentamicin, amikacin), ß-lactam antibiotics (amoxicillin, ampicillin, cefuroxime axetil, and ceftriaxone), macrolides (erythromycin, azithromycin, clarithromycin), clindamycin, and trimethoprim-sulfamethoxazole (90, 122).

Doxycycline has the additional benefit of being highly active against B. burgdorferi. Longer courses of doxycycline therapy are required for treatment of LD coinfection, with a 14-day course for early localized infection with B. burgdorferi (EM) and longer regimens (28 days) for disseminated or late complications. Other antibiotics commonly used in treating early and late LD among both pediatric and adult populations (e.g., amoxicillin and ceftriaxone) are ineffective in treating HA. In contrast, doxycycline not only is highly active against both A. phagocytophilum and B. burgdorferi but also has the advantage of being active against other tick-borne infections, such as ehrlichiosis, Rocky Mountain spotted fever, other spotted fever group rickettsioses, and tick-borne relapsing fever (caused by Borrelia species). For patients with HA who have clinical or laboratory features indicative of coinfection with B. burgdorferi, a longer course of doxycycline (14 to 21 days) is recommended, according to evidence-based treatment recommendations for LD presented by the Infectious Diseases Society of America (223). The ultimate choice and duration of antimicrobial therapy should be guided by the stage of LD. Clinicians might want to empirically treat HA patients from regions of LD endemicity with 14 days of doxycycline, because B. burgdorferi coinfection is difficult to rule out during the acute illness phase, and it has been reported for as many as 10% of HA patients (128).

More recently, combination therapy with atovaquone (for adults, 750 mg every 12 h with meals; for children, 20 mg/kg every 12 h with meals) and azithromycin (for adults, 500 mg on day 1 and 250 mg/day thereafter; for children, 12 mg/kg/day) for 7 days has been demonstrated to be as effective as 7 days of combination therapy with clindamycin-quinine for patients with non-life-threatening B. microti infections (103, 124). Higher doses of azithromycin (500 to 1,000 mg daily) and 10 days of total combination therapy with atovaquone have been employed also (124, 219). Fifteen percent of patients on atovaquone-azithromycin reported adverse reactions, most commonly diarrhea and rash; clindamycin-quinine was associated with adverse reactions (tinnitus, diarrhea, and decreased hearing) for 72% of patients (103).

Partial or complete exchange transfusions have been used for severely ill babesiosis patients with substantial hemolysis, renal or pulmonary compromise, or high levels of parasitemia (10%) (26, 78, 101). When given concurrently with appropriate chemotherapy, whole-blood exchange transfusion might be life-saving (58). Human infections with the bovine pathogen B. divergens should also be regarded as a medical emergency, and prompt therapy is indicated. Exchange transfusions in combination with intravenous clindamycin and intravenous or oral quinine have been successful in treating B. divergens in Europe (26, 27).

Certain antiprotozoal and antimalarial therapeutic regimens have been largely unsuccessful, including chloroquine, primaquine, pyrimethamine, pyrimethamine-sulfadoxine, tetracycline, minocycline, pentamidine isethionate, and trimethoprim-sulfamethoxazole (100, 101). Clinicians should be aware that treatment for babesiosis will not provide effective treatment for LD or HA. Neither clindamycin, quinine, atovaquone, nor azithromycin provides any activity against B. burgdorferi or A. phagocytophilum. Under these circumstances, patients with B. microti/divergens infections who are suspected of also having LD or HA require an additional antimicrobial (e.g., doxycycline) (131).

STRATEGIES FOR PREVENTING COINFECTIONS FROM IXODES TICKS

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Prevention of LD and other diseases transmitted by Ixodes ticks has posed a substantial public health challenge. The majority of prevention programs stress personal protection measures such as tick habitat avoidance (including information on when and where people are at risk), protective clothing, repellents, and frequent tick checks to locate and remove ticks before they can transmit disease. Although these efforts are useful, completely avoiding tick habitats, maintaining regular repellent use, wearing protective clothing in warm weather, or locating attached ticks (especially smaller nymphs) often can be impractical or impossible (157). Research demonstrating the efficacy of personal protection measures is lacking, and tick-borne disease case numbers continue to increase in parts of the world where such diseases are endemic (79).

While not routinely recommended, a single-dose prophylactic antibiotic treatment (e.g., doxycycline) of known tick bites to prevent potential LD is often practiced in the United States. At present, tick bite antibiotic prophylaxis is the only available preventive measure proven effective in preventing LD (132). However, doxycycline should be taken only by individuals who have had a recognized Ixodes tick bite within the previous 72 h, and where there is reasonable information to suggest that the tick remained attached long enough (24 to 48 h) to potentially transmit B. burgdorferi if it was infected. Medical