Zoonotic Babesia: Possibly emerging pathogens to be considered for tick-infested humans in central Europe

Int. J. Med. Microbiol. 293, Suppl. 37, 93-103 (2004) © Urban & Fischer Verlag

IJ M

http:llWWW.urbanfischer.deljournalslijrnrn

Mini-Review Zoonotic Babesia: Possibly emerging pathogens to be considered for tick-infested humans in central Europe K.-P. Hunfeld, V. Brade Institute of Medical Microbiology, University Hospital of Frankfurt, Germany

Abstract The three host-tick Ixodes (I.) ricinus is regarded as an important vector of tick-borne microorganisms pathogenic for humans in central Europe and is primarily known as the main vector of Borrelia (B.) burgdorferi and the virus causing tick-borne encephalitis (TBE), the most clinically relevant tick transmitted pathogens for humans in European countries. Furthermore, it is now well established that I. ricinus also transmits Ehrlichia (E.) phagocytophila, Babesia (Ba.) divergens, and Ba. microti, all agents of zoonotic infections in dear, sheep, cattle, dogs, and horses. In addition to their known zoonotic potential, recent molecular-epidemiological and seroepidemiological surveys as well as increasingly reported clinical cases of infections caused by these tick-borne organisms other than B. burgdorferi (TOBB) also strongly suggest a possible relevance of Babesia, Ehrlichia and Rickettsia for humans at risk in Europe. However, there are few medical microbiological investigations and epidemiological data on the distribution and relevance of Babesia for humans in our part of the northern hemisphere. There is also very little diagnostic and clinical knowledge on human babesiosis in many regions of Europe. Furthermore, sophisticated diagnostic tools designed for the reliable detection of the underlying pathogens, are not yet generally available to the microbiological laboratory. This review aims to provide basic information on human babesiosis and the most relevant causative pathogens of the disease in Europe and to draw attention to this parasitic infection as a possibly emerging and probably underdiagnosed disease in this part of the northern hemisphere. Key words: Babesiosis - humans - review - taxonomy - diagnostics - epidemiology - clinics - Europe

Introduction Tick-borne infectious diseases are among the most interesting and potentially devastating infections of mankind and animals and there is a steady worldwide increase in both the recognition and incidence

of infections derived from ticks (Cunha, 2000). Historical sources dealing with tick-associated pathogens in animals can be traced back to the book "Exodus" of the Holy Bible. The plague ( "murrain" ) visited upon the cattle of Pharao Ramses H is

Corresponding author: Klaus-Peter Hunfeld, Institut fiir Medizinische Mikrobiologie, J. W. Goethe-Universit~it, PaulEhrlich-Str. 40, 60596 Frankfurt, Germany, Phone: 00496963016441, Fax: 004969630183066, E-maih [email protected] 1433-1128/04/293/Suppl.37-93 $15.00/0

94

K.-P. Hunfeld, V. Brade

probably the first historical reference to babesiosis (Gelfand, 2000). However, it took until the late 18 th century to prove the existence of tick-borne microorganisms by scientific evidence and to reach better understanding of the life cycles of the pathogens and the circumstances of their transmission. In 1893 Smith and Kilbourne discovered that the causative agent of Texas cattle fever, now known as the protozoan Babesia (Ba.) bigemina, is transmitted by ticks. They were the first to introduce these arthropods as important vectors of anthropozoonoses (Smith and Kilbourne, 1893). The importance of ticks in the transmission of disease was further substantiated by McCalla and Brereton in 1908 in the USA; a tick obtained from a patient suffering from rocky-mountain spotted fever was used to transmit the disease to two healthy volunteers (Weis, 1988). In 1909 Rickett discovered the eponymous genus of bacteria responsible for Rocky Mountain spotted fever - Rickettsia (Ricketts, 1909). Until now, up to 27 tick-borne pathogens (Table 1), parasites, bacteria and viruses have been identified that may be of considerable concern for tick infested humans in Germany and other parts of central Europe (Faulde etal., 2001). Many more are expected to follow soon. These infections constitute a broad spectrum of, what are in some cases, complicated and dangerous diseases that differ in their vectors and clinical manifestations and, therefore, can be easily confused with a wide variety of infectious and non-infectious diseases resulting in difficult differential diagnostic considerations. Moreover, tick-borne infectious diseases affect both, livestock and humans all over the world. Although, much about tick-conducted anthropozoonoses still remains puzzling, no doubt remains that these pathogens cause enormous costs to the veterinarian and human public health systems. For Lyme borreliosis (LB) the tremendous dimension of the problem is underlined by a 1998 pharmacoeconomic analysis sponsored by SmithKline Beecham (Philadelphia, USA) which estimated the direct and indirect costs of LB alone in the United States, including costs of therapeutic treatments, to be $ 2.5 billion over 5 years (Maes et al., 1998). Likewise, with regard to babesiosis one has to imagine that in South Africa alone treatment of canine babesiosis costs the dog owning public more than R 20 million each year (Collett, 2000). Thus, tick-borne pathogens clearly represent an important public health threat worldwide. Prevention and recognition of tick-transmitted infections, therefore, depicts a significant challenge to clinicians and public health professionals. To date, the epidemiology, geographic distribution, and medical

importance of many newly recognised tick-borne pathogens including Babesia and Ehrlichia, the recently emerged agents of human ehrlichiosis and human babesiosis, however, remain uncertain and have to be definitively established in many parts of the world and especially in Europe (Hunfeld and Brade, 1999; Hunfeld et al., 2002a).

Emergence of tick-transmitted diseases In central Europe Ixodes (I.) ricinus is primarily known as the main vector of Borrelia (B.) burgdorferi and tick-borne encephalitis which together are estimated to infect some 100,000 individuals every year (Scheibe et al., 1998; Hunfeld et al., 1998a). B. burgdorferi is considered as the most clinically relevant tick-transmitted pathogen in the European countries. With an estimated number of 30,000 to 60,000 new cases per year in Germany alone, LB is now regarded as the most common tick-borne zoonosis in Germany (Scheibe et al., 1998; Wilske et al., 2000) In contrast, infections secondary to Babesia and Ehrlichia are commonly accepted as causing veterinary disease in central Europe. Whether these microorganisms ought to be regarded pathogens of considerable medical importance for humans in many European countries as well, to date, requires further investigation (Hunfeld and Brade, 1999). Nevertheless, babesiosis, which remains one of the more common diseases of flee-living animals and livestock worldwide, has currently been rediscovered as a possibly emerging tick-borne zoonosis in humans, too (Bronsdon et al., 1999; Krause et al., 1999; Kjemtrup et al., 2000; Homer et al., 2000; Hunfeld et al., 2002a, 2002b; Foppa et al., 2002). Since the late 1950s two species of Babesia in particular, i.e. the cattle species Ba. divergens in Europe and the rodent species Ba. microti in North America, have been shown to cause a significant number of infections in humans (Herwaldt et al., 1996; Granstr6m et al., 1997, Kjemtrup etal., 2000). During the last decade increasing numbers of human infections with in part newly recognised Babesia spp. obviously pathogenic for humans have been reported especially from the northern and western parts of the USA but also abroad (Homer et al., 2000; Kjemtrup et al., 2000). Subsequently, in several European countries studies based on polymerase chain reaction (PCR) and DNA sequence analyses also have substantiated that tick-borne pathogens other than B. burgdorferi (TOBB), like Ehrlichia, Rickettsia, and Babesia, are widely pre-

Babesiosis in humans

95

Table 1. Vectors and tick-borne pathogens of considerable concern for humans in Germany and other parts of central Europe (modified from Faulde et al., 2000). Pathogen

Target

Disease

Vector

Viruses Flaviviridae TBE virus Coltiviridae Eyach virus

Systemic disease, CNS

Tick-borne encephalitis

L ricinus, L persulcatus

Systemic disease, CNS

Neurological disease, Eyach virus-fever

L ricinus

Nairoviridae Ewe virus

Systemic disease, CNS

Neurological disease, Erve virus-fever

L ricinus, Dermacentor (D.) rnarginatus

Reoviridae Tribec virus

Systemic disease, CNS

Neurological disease, Meningoencephalitis (?)

L ricinus

Reoviridae Lipovnik virus

Systemic disease, CNS

Meningoencephalitis (?)

L rMnus, Rhipicephalus (Rh.) sanguineus

Bunyaviridae Uukuniemi virus

Systemic disease, CNS

Encephalitis, neurological disease

L ricinus

Unclassified Bhanja virus

Systemic disease, CNS

Bhanja virus-fever, Meningoencephalitis

L ricinus, D. marginatus

Bacteria Obligate intracellular bacteria Rickettsiae R. conorii R. helvetica R. sibirica R. mongolotimonae R. slovaca

Endothelial Endothelial Endothelial Endothelial Endothelial

Mediterranean spotted fever

Rh. sanguineus L ricinus, D. marginatus Dermacentor spp. unknown L ricinus, L persulcatus, D. marginatus

Coxiellae C bumetii

Systemic disease

Q fever

Rh. sanguineus, L ricinus, D. marginatus

Ehrlichiae E. equi

Granulocytes

L dcinus, I. persu/catus

E. phagocytophila

Granulocytes

E. canis

Granulocytes

Granulocytic ehrlichiosis of animals and humans Granulocytic ehrlichiosis of animals and humans Monocytic ehrlichiosis of canides and humans Lyme borreliosis Lyme borreliosis Lyme borreliosis Lyme disease (?)

I. L L L

Tularemia

L ricinus, D. marginatus

Babesiosis Babesiosis Babesiosis Babesiosis

L ricinus L ricinus I. ricinus L ricinus

Spirochetes B. burgdorferi s.s. B. garinii B. afzelii B. valaisiana Francisellae F. tularensis Parasitic agents Babesiae Ba. divergens Ba. bovis Ba. microti Ba. odocoilei

cells cells cells cells cells

Systemic disease Systemic disease Systemic disease Systemic disease (?)

Erythrocytes Erythrocytes Erythrocytes Erythrocytes

Siberian tick typhus Spotted fever Tick-borne lymphadenitis (TIBOLA), spotted fever

valent in the three-host tick I. ricinus (Johansson et al., 1995; Schouls et al., 1999; Nilsson et al., 1999; Baumgarten et al., 1999; Duh et al., 2001; Skotarczak et al., 2001; Foppa et al., 2002), whose larvae, nymphs, and adults feed on different hosts, including virtually any warm-blooded animal and

L dcinus, L persu/catus Rh. sanguineus, D. marginatus (.2) ricinus, ricinus, ricinus, ricinus,

L persulcatus L persu/catus, L persulcatus, L persulcatus,

humans. This tick is known as the most common human questing tick in western and central Europe (Scheibe et al., 1998). Moreover, with regard to babesiosis recent seroepidemiological surveys as well as increasingly reported clinical cases strongly suggest a possible relevance of Babesia spp. for

96

K.-P. Hunfeld, V. Brade

humans at risk also in Europe (Popovsky et al., 1991; Herwaldt et al., 1996; Hunfeld et. al., 1998a; Krause et al., 1999; Kjemtrup et al., 2000, Hunfeld et al., 2002b). Nevertheless, in many of these regions microbiological investigations and the clinical knowledge of human disease secondary to babesiae is low and more sophisticated diagnostic tools designed for the reliable detection of the underlying pathogens, are not yet widely available. As a matter of fact, detailed epidemiologicalinformation and the true number of cases of human babesiosis in most parts of Europe, therefore, have still to be determined exactly. Consequently, human babesiosis, although possibly emerging, probably represents an underdiagnosed disease in this part of the northern hemisphere.

Epidemiology of human babesiosis in Europe In the past, human babesiosis has been considered to occur rarely in Europe, with only about 30 reported cases, and all but four clinical cases have been attributed to Ba. divergens, which has been diagnosed mainly in splenectomised patients in former Yugoslavia, in France and Great Britain (Gorenflot et al., 1998; Homer et al., 2000; Gelfand, 2000). In these cases, the patients presented with acute febrile hemolytic disease and their clinical courses have almost always been fatal. Reports on human infections with Ba. microti in Europe have been sparse, and little is known about their frequency and importance in that part of the northern hemisphere (Uhnoo et al., 1992; Granstr6m, 1997; Sharan et al., 2000; Hunfeld et al., 2002a). The frequent reports of cases of human babesiosis in North America are in contrast to only sporadic reports of the disease in Europe and other parts of the world. However, in many non-American regions microbiological investigations and awareness of the clinical presentation of babesiosis in humans lag far behind those in North America. Moreover, these data are unlikely to accurately reflect the true epidemiological situation of Babesia infections or distribution of the pathogens in the European human population. Rather, it is likely that increasing scientific and medical interest in human babesiosis world-wide will result in larger numbers of reported cases in Europe and other parts of the world, and different clinical pictures of the disease in immunocompetent hosts will emerge (Homer et al., 2000). Interestingly, both Ba. microti and Ba. divergens are known to occur in ticks, livestock, and rodents

(Clethrionomys glareolus, Microtus agrestis) in Germany (Walter et at., 1984; Mehlhorn et al., 1986; Huwer et al., 1994, Hunfeld et al., 2002a). The possible relevance of Babesia spp. for tickexposed individuals in European countries was very recently substantiated further by demonstrating the presence of Ba. microti, Ba. divergens, and closely related species in I. ricinus ticks in Slovenia and Poland by PCR (Duh et al., 2001; Skotarczak et al., 2001). In Germany, this tick is regarded as the main vector of tick-borne infections and is widely distributed (Scheibe et al., 1999). Recent seroepidemiological studies suggested that infections with Babesia spp. may occur more frequently than previously believed in tick-exposed patients in Europe, with seroprevalences ranging between 4 and 13 % in the investigated populations (Granstr6m, 1997). For Ba. rnicroti, sporadic reports from France and Germany have claimed asymptomatic seropositives, but few systematic studies have been conducted there as yet (Gorenflot et al., 1987; Uhnoo et al., 1992; Granstr6m, 1997; Hunfeld et al., 1998a). To further contribute to the ongoing discussion of whether or not infections with Babesia spp. are common in the German human population, recently we have used antigens of Ba. microti and Ba. divergens in a seroepidemiological study to investigate under more optimised conditions the frequency of infections with Babesia spp. in tick-exposed patients and human control groups in midwestern Germany as surrogate markers for the detection of antibodies directed against species of Babesia (Hunfeld et al., 2002b). The prevalence of antibodies to either Ba. microti or Ba. divergens in tick-exposed individuals was significantly higher (11.5 %) than in the control group of healthy blood donors (1.7%). Interestingly, titres of _>1 : 256 against at least one of the babesial antigens, possibly indicating a more recent infection with the pathogen (Hunfeld et al., 1998; Gelfand, 2000), were found with significantly greater frequency (9 versus 1, p < 0.05) in tickexposed patients (n = 225) than in the other control groups (n=242) (Hunfeld et al., 2002b). This is interesting to note because the seroprevalence of infections with Babesia spp. in the tick-exposed humans as observed in these studies is similar to that found in companion animals in Germany. Thus, a recent seroepidemiological study reported a seroprevalence of 15% for Ba. microti in dogs in Germany (Metz, 1999). Any discussion of seroepidemiological data concerning babesiosis, however, must take into account interspecies reactivity of antigenic components within the genus Babesia and cross-reactivity with

Babesiosis in humans other bacterial or parasitic agents (Hunfeld et al., 1998a; Homer et al., 2000; Kampen et al., 2002). Our results obtained with sera from patients with active or recent toxoplasmosis, malaria, or syphilis did not demonstrate increased reactivity in the IFA tests used (Hunfeld et al., 2002b). If cross-reactions between Babesia and Borrelia were to occur, one would expect corresponding interactions with Treponema pallidurn, to which B. burgdorferi shows a close antigenic relationship (Hunfeld et al., 1998a; Hunfeld et al., 2002b). Moreover, the seroprevalences of Babesia infections in tick-exposed patients and in blood donors found here correlate well with those of a study of Swedish Lyme borreliosis patients, in which 13% of the individuals were found to be seropositive for Ba. divergens (Uhnoo et al., 1992), and with a study in western France that indicated 0.5% asymptomatic seropositives out of 408 individuals investigated when a more conservative cut-off of > 1:80 was used (Gorenflot et al., 1987). Moreover, the overall prevalence of antibodies to babesial antigens (24 out of 225 samples: 11.5 %) in the tick-exposed individuals tested in our recent study is similar to the positivity rate for Babesia in ticks (9.6 to 16.3%) as recently determined by molecular biological methods in Europe (Duh et al., 2001; Skotarczak et al., 2001). As outline above, although probably prevalent in ticks all over Europe almost nothing is known, however, about its epidemiology and possible medical bearing for humans in most parts of central and Eastern Europe. Consequently, further seroepidemiological and molecular epidemiological studies are required to learn more about the true distribution and medical relevance of babesial pathogens in the various parts of Europe.

Taxonomy, genetic diversity and microbiological properties of the genus Babesia Babesiosis is caused by intraerythrocytic parasites of the protozoan genus Babesia. The parasites are 0.5 to 2 ~rn in size (Fig. 1) and are named after the Romanian scientist Victor Babes, who in 1888 first identified pear-shaped, Plasmodium-like protozoan parasites as a cause of febrile hemoglubinuria in cattle (Babes, 1888). The genus Babesia is phylogenetically related to other sporozoan genera like that of Toxoplasma and Plasmodium. To date, about 100 species of Babesia, phylum Apicomplexa have been described worldwide, based on intraery-

97

throcytic stages detected in mammals (Levin et al., 1988). However, their pleomorphism in different species of mammalian hosts and the results of recent molecular systematic studies render some of these descriptions dubious. It is now assumed that some species of Babesia are less host-specific than previously believed and that the number of valid species of Babesia will be gradually reduced as more information becomes available on them (Cox et al., 1998; Krause et al., 1999; Homer et al., 2000). Since the late 1950s, two species of Babesia in particular, i.e. the cattle species Ba. divergens in Europe and the rodent species Ba. microti in North America, have been shown to cause a significant number of infections in humans (Granstr6m, 1997; Kjemtrup et al., 2000). In addition, other Babesia spp. of unknown identity currently designated as WAl-type, MO1, and CA1 to CA4, have been found to be pathogens of considerable concern for humans in the USA (Popovsky et al., 1991; Herwaldt et al., 1996; Krause et al., 1999; Kjemtrup et al., 2000), with recent molecular phylogenetic studies suggesting that some of these parasites may be derived from dogs or wildlife (Kjemtrup et al., 2000). The most important species considered pathogenic for humans in Europe are summarized in Table 2. It has also been suggested that species of Babesia, formerly described as belonging to the genus Entopolypoides, infecting rhesus monkeys may have zoonotic potential (Bronsdon et al., 1999). While the specific vectors of many Babesia spp. are still unknown, those of zoonotic potential are commonly accepted to be transmitted to their vertebrate hosts by ixodid ticks (Krause et al., 1999; Homer et al., 2000; Kjemtrup et al., 2000). I. ricinus is also regarded as the most important vector for tickborne diseases in humans in these regions and, although not known for certain, there is circumstantial evidence that I. ricinus transmits at least Ba. divergens and Ba. microti to humans (Cox et al., 1998; Granstr6m, 1997, Gray et al., 2002; Hunfeld et al., 2002a, b). Consequently, it has been suggested that babesiosis in humans is an underdiagnosed disease in the European part of the northern hemisphere (Granstr6m, 1997; Gray et al., 2002; Hunfeld et al., 2002a).

Clinical disease manifestation Babesiosis in the USA has varied substantially from that described in Europe. Several hundreds of infections with Ba. microti in humans have been

98

K.-P. Hunfeld, V. Brade

Table 2. Babesia spp. of medical relevance for human disease in Europe (adapted from Gorenflot et al., 1998). Species

Reservoir

No. of cases Distribution (Europe)

Ba. divergens

Cattle

22

Ba. microti

Small mammals 2

Ba. boris

Small mammals Cattle Small mammals Dogs Small mammals Deer

Ba. canis

WA-1 Ba. odocoilei

2 1 unknown 2

Europe, Africa, Asia Northern Hemisphere Northern Hemisphere Northern Hemisphere USA, Europe (?) Europe, USA

reported from the coastal areas of the New England states of the USA (Homer et al., 2000; Kjemtrup et al., 2000). The clinical spectrum of the infection ranges from asymptomatic to rapidly progressive and fatal (Table 3). Although babesiosis can affect persons of all ages, most of the patients present in their 40s or 60s (Gorenflot et al., 1998; Scharan et al., 1999; Mylonakis, 2000). The peak transmission months are from May to September. The incubation period varies from five to 33 days after a tick bite, but most individuals do not remember a tick infestation (Gorenflot et al., 1998; Scharan et al., 1999). After an infected blood transfusion the incubation period can be up to nine weeks (Mylonakis, 2000). Clinical symptoms of babesiosis include high fever (up to 40 °C), chills, diaphoresis, anemia, weakness, fatigue, anorexia, and headache. Later on, the patient may develop, jaundice, dark urine, central nervous system involvement or severe complications like congestive heart failure, intravascular coagulation renal failure, and respiratory distress syndrome (Herwaldt etal., 1991; Gorenflot etal., 1998; Scharan etal., 1999; Mylonakis, 2000; Homer et al., 2000; Kjemtrup et al., 2000). In a recent retrospective analysis of 139 hospitalised patients in New York State 25% of the individuals required hospitalisation and 6.5 % died. On average, after the onset of symptoms a 12 to 14-day delay was noted before initiation of appropriate antibiotic treatment (White et al., 1998). The phenomenon of co-infections with Babesia and other tick-borne pathogens, particularly with B. burgdorferi, has caused growing concern. In Europe there are only a few reports on potential co-infection with Ba. divergens, as determined by seroreactivity (asymptomatic infection), and B. burgdorferi (Gorenflot et al., 1998; Hunfeld et al., 2002b). In serosurveys from Babesia-endemic areas in the USA,

however, it is estimated that as many as 13% of the Lyme disease (LD) patients are co-infected with Ba. microti (Krause et al., 1992; Krause et al., 1996; Gorenflot et al., 1998; White et al., 1998). Furthermore, it has been speculated that the increasing Ba. microti seropositivity seen during the past 30 years in the USA is consistent with the increased incidence of LD (Benach etal., 1981; Homer etal., 2000). Patients coinfected with babesiae experience more severe symptoms (Krause et al., 1996). Very recently, the first two human cases of babesiosis in Italy and Austria, which occurred in two asplenic men, were reported. In phylogenetic analysis, the causative pathogen clusters with Ba. odocoilei, a parasite of white-tailed deer, and these two organisms form a sister group with Ba. divergens (Herwaldt et al., 2003). When dealing with the actual frequency of infections in European countries, it must be borne in mind, however, that in most immunocompetent individuals suffering from babesiosis the disease is probably mild and self-limiting. Likewise, differences in the pathogenicity of strains endemic in different geographic areas may explain the variable clinical disease manifestations (Hunfeld etal., 2002a). In addition, the prevalence of Babesia spp. in ticks is known to vary significantly even on a more local geographic level (Foppa et al., 2002). In the immunocompetent host it is likely that most cases take a subclinical course and that undiagnosed carriers do exist (Homer et al., 2000; Hunfeld et al., 2002a). Moreover, differential diagnostic separation from LB can be difficult because the initial symptoms of both human babesiosis and Lyme borreliosis are known to overlap significantly (Table 3), causing non-specific symptoms such as fever, fatigue, and flu-like illness (Granstr6m, 1997; Sharan et al., 2000; Mylonakis et al., 2001). Thus, patients with inadequate response to appropriate therapy of proven or suspected LB following a tick bite should be examined for infections with other tick-borne agents, including human granulocytic Ehrlichia spp., TBE, and Babesia spp. (Hunfeld et al., 2002). The apparent existence and high prevalence of chronic babesial infections may become increasingly important because, although rare, asymptomatic but chronically infected blood donors are now known to be a source of transfusiontransmitted babesiosis in areas where Babesia spp. with zoonotic potential are endemic (Popovski, 1991). In a study from Connecticut the risk of acquiring babesiosis from a transfused pack of red blood cells was estimated at about 0.17% but was lower from a transfused unit of platelets (Gerber et al., 1994).

Babesiosis in humans

99

Table3. Symptoms associated with LD and babesiosis modified from (Homer et al., 2000)

% of patients exhibiting the indicated symptoms Symptom

LD (n =214)

Babesiosis Both infections (n=10) (n =26)

Fatigue Headache Erythema migrans (EM) Fever Sweats Chills Myalgia

49 42 85 42 11 23 31 14 36 5 21 14 10 9 3 0 4 3

60 60 0 80 20 50 20 10 50 10 30 0 20 20 0 10 0 0

Anorexia Arthralgia Nausea Neck stiffness Multiple EM Cough Sore throat Conjunctivitis

Splenomegaly Vomiting Joint swelling

81 77 62 58 46 42 38 31 27 23 23 19 15 15 12 8 8 4

Microbiological diagnostics Non-specific laboratory findings in clinically apparent cases may show high levels of transaminases, alkaline phosphatases, unconjugated bilirubin, and lactic dehydrogenase. Normochromia, normocytic anemia, thrombocytopenia, and occasionally, leucopenia may also be present probably due to tumor necrosis factor (TNF)-mediated immune response similar to that seen in severe cases of malaria (Krause et al., 1999; Gelfand, 2000; Homer et al., 2000; Sharan et al., 2000; Mylonakis et al., 2001). Up to now, the microbiological diagnosis of human babesiosis in Europe has mainly been based on the detection of the parasites in Giemsa-stained blood smears (Fig. 1) of patients with clinical symptoms of disease. Although rapid diagnosis can be made with thin blood smears, parasites are often not visualised early in the course of infection or owing to low levels of parasitemia in the non-immunocompromised host (Krause et al., 1999; Gelfand, 2000; Homer et al., 2000; Sharan et al., 2000; Mylonakis et al., 2001). Currently, the gold standard for confirming suspected cases of Babesia infection is the intraperitoneal inoculation of 1.0 ml EDTA-whole blood in golden hamsters or gerbils (Krause et al., 1999; Gelfand, 2000; Homer et al., 2000; Sharan et al., 2000). Within 2 to 4 weeks, smears will be positive in the infected animal. PCR (Fig. 2) is a new rapid

Fig. 1. Microscopic appearance of Babesia in Giemsa stained blood smears. Erythrocytes infected with Ba. microti parasites (arrows), magnification 1000× oil.

diagnostic technique for the detection of Babesia spp. that has not yet been standardised and systematically evaluated. First results, however, are promising and revealed PCR to be a sensitive and specific tool in the diagnosis of acute babesiosis (Eskow et al., 1999). Indirect detection of the pathogen is frequently performed by IFA testing for IgG and IgM antibodies (Fig. 2). IFA tests are known to be sensitive, specific, and reproducible for diagnosis of Ba. microti-specific antibodies in human sera (Gelfand, 2000; Homer et al., 2000; Sharan et al., 2000; Krause et al., 1994). Multicenter studies dealing with the reliability of Ba. microti IFA in the diagnosis of human babesiosis reported specificities ranging from 90 - 100% with comparable test systems. Moreover, titres from 1 : 32 to 1 : 160 were reported to be both diagnostic and specific, with positive predictive values of 69 - 100% and negative predictive values of 9 6 - 9 9 % (Krause et al., 1994). One drawback of these tests, however, is that the production of antibodies takes up to 14 days after infection. Consequently, many patients remain seronegative at the onset of clinical symptoms. Problems with diagnosing Babesia infections in humans hide the fact that diagnostic tools such as indirect fluorescent antibody (IFA) tests or polymerase chain reaction (PCR)-based assays (Fig. 2) designed for specific and reliable detection of the pathogens, are not readily available to diagnostic laboratories in Europe. In addition, the use of ELISA testing, confirmative immunoblotting or genusspecific PCR techniques for diagnostic purposes, to date, has not been sufficiently evaluated for the use of such tests in the routine microbiological laboratory.

100

K.-P. Hunfeld, V. Brade

Fig. 2. (A) Indirect immunofluorescence assay (IFA): Babesia reactive serum sample (titre 1:128), multiple fluorescing spots (Ba. microtl) in fixed gerbil erythrocytes. (B) Indirect immunofluorescence assay (IFA): Babesia reactive serum sample (titre 1:256), multiple fluorescing spots (Ba. divergens) in fixed gerbil erythrocytes. (C) PCR directed against a 238-bp gene fragment of Bo. microtL M: marker (123 bp marker), S: samples.

conventional chemotherapy is reserved for those individuals who are extremely ill, i.e. with massive hemolysis, asplenia, immunosuppression and blood parasitemia of more than 10% (Mylonakis et al., 2001). Although, in Europe there are only few reports of potential co-infection with Ba. divergens the phenomenon of co-infections of tick infested patients with Babesia and other tick-borne pathogens, particularly with B. burgdorferi, has caused growing concern. As parasitic agents Babesia spp. are not susceptible to common antibiotics like doxycycline or amoxycillin which are frequently used in the treatment of LB. Therefore, further evaluation of the susceptibility profile of these pathogens is of interest but remains difficult due to a lack of standardised in vitro testing methods (Brasseur et al., 1998). Should new broad spectrum antimicrobial agents prove effective against Babesia spp., they may serve as useful agents in a chemotherapeutic approach that tries to cover several of the newly emerged tick-borne pathogens, including E. phagocytophila, Rickettsia spp., and Babesia spp.

Treatment and antimicrobial susceptibility patterns

Conclusion

Most infections due to Ba. microti show mild to subclinical disease manifestation and resolve on their own without antimicrobial chemotherapy. Individuals with Ba. divergens infection, most of which are immunocompromised or asplenic, must be regarded as medical emergencies and require immediate treatment including chemotherapy and possibly exchange transfusion to arrest hemolysis and to prevent renal failure. Several drugs have been tested in vitro but results for tetracycline, primaquin and pentamidin have varied. Imidocarb was highly effective in vitro but has not been approved for use in humans (Brasseur et al., 1998; Mylonakis et al., 2001). The anti-malaria drug chloroquin is not effective against Babesia. Treatment of human babesiosis with a combination of quinine and clindamycin for seven to 10 days is the most commonly used treatment. A recent prospective, randomised study in humans compared the efficacy of this treatment regimen with the clinical outcome of atovaquon and azithromycin in cases of Ba. microti infection. Although the clinical outcome was similar between the two patient groups the application of atovaquon plus azithromycin lowered the relative number of side-effects to 15% versus 72% in the group of patients receiving quinin and clindamycin (Krause et al., 2000). The treatment of human babesiosis by exchange transfusion in addition to

In central Europe B. burgdorferi and TBE virus remain the most clinically relevant tick transmitted pathogens for humans. In addition to these established human disorders, infections caused by TOBB including Babesia spp. and Ehrlichia spp. have gained increasing attention. Significant progress has been made over the past decades in the recognition and the better understanding of tick-borne diseases resulting in increasing awareness that tickborne infections owing to TOBB may represent an important public health problem in many parts of Europe. Nevertheless, up to now, much about these vector-borne anthropozoonoses still remains puzzling. Recent seroepidemiological studies on tickinfested individuals as well as several molecular biological investigations on Ixodes ticks provide increasing evidence that infections due to Babesia spp. may occur more frequently in tick-infested humans in central Europe than previously believed. Yet another possible explanation for the variability of symptoms expressed by some patients after tick infestation, therefore, might be undiagnosed infection or coinfection with one of the above-mentioned pathogens (Table 1) or other as yet unknown infectious agents. Recognition of infections like human babesiosis represents a significant challenge to clinicians and public health professionals as it largely relies on good clinical knowledge of the

Babesiosis in humans differential diagnosis of tick-borne diseases and on microbiological testing in specialist laboratories. A problem that remains to be solved is that diagnostic tools and well evaluated assay systems designed for specific and reliable detection of these pathogens are not readily available to diagnostic, microbiological laboratories in Europe. Moreover, knowledge about the true distribution and medical relevance of tickborne pathogens like Babesia spp. remains sparse for many parts of Europe and should rapidly be increased by gathering additional epidemiological data. For patients exposed to ticks in areas where multiple tick-borne pathogens are endemic, it seems reasonable for clinicians to be aware of clinical signs caused by TOBB that may be consistent with each infection alone or in combination, especially, when patients with LB or individuals with fever and nonspecific symptoms after tick infestation fail to respond promptly to antibiotic therapy. Moreover, although rare, infections due to TOBB like Babesia spp. should always be considered in the differential diagnosis of fever of unclear origin in the increasing number of i m m u n o c o m p r o m i s e d patients and in travellers returning from countries outside central Europe. In the meantime, although doxycycline remains the drug of choice for the first-line treatment of tick-borne diseases in nonpregnant patients > 8 years of age, azithromycin should be borne in mind for patients where early LB and concurrent babesiosis (Table 3) are considered in the differential diagnosis of a febrile illness following a tick bite.

References Babes, V.: Sur l'hemoglobinurie bacterienne du boeuf. Compt. Rend. Acad. Sci. 107, 692-694 (1888). Baumgarten, B.U., R611inghoff, M., Bogdan, C.: Prevalence of B. burgdorferi and granulocytic and monocytic ehrlichiae in I. ricinus ticks from southern Germany. J. Clin. Microbiol. 37, 400-403 (1999). Benach, J. L., Habicht, G. S." Clinical characteristics of human babesiosis. J. Infect. Dis. 144, 481 (1981). Brasseur, P., Lecoublet, S., Kapel, N., Favennec, L., Ballet, J. : In vitro evaluation of drug susceptibilities of Babesia divergens isolates. Antimicrob. Agents Chemother. 42:818 - 820 (1998). Bronsdon, M.A., Homer, M.J., Magera, J.M.H., Harrison, C., Andrews, R. G., Bielitzki, J. T., Emerson, C. L., Persing, D. H., Fritsche, T. R.: Detection of enzootic babesiosis in baboons (Papio cynocephalus) and phylogenetic evidence supporting synonymy of the genera Entopolypoides and Babesia. J. Clin. Microbiol. 37, 1548-1553 (1999). Burgdorfer, W., Barbour, A. G., Hayes, S.F., Benach, J. L., Grunwaldt, E., Davis, J. P." Lyme disease - a

101

tick-borne spirochetosis? Science 216, 1317-1319 (1982). Collett, M.G.: Survey of canine babesiosis in South Africa. J. S. Aft. Vet. Assoc. 71, 180-186 (2000). Cox, F. E. G." Babesiosis and malaria. In: Palmer, S. R., Soulsby, E. J. L., Simpson, D. I. H.: Zoonoses: biology, clinical practice, and public health control. Oxford University Press, Oxford, pp. 599-607 (1998). Cunha, B. A.: Tick-borne infectious diseases: diagnosis and management. Marcel Decker Inc., New York, pp. V-VI (2000). Duh, D., Petrovec, M., Avsic-Zupanc, T.: Diversity of babesia infecting European sheep ticks (Ixodes ricinus). J. Clin. Microbiol. 39, 3395-3397 (2001). Eskow, E. S., Krause, P.J., Spielman, A., Freeman, K., Aslanzadeh, J.: Southern extension of the range of human babesiosis in the eastern United States. J. Clin. Microbiol. 37, 2051-2052 (1999). Faulde, M., Hoffmann, G.: Vorkommen und Verhiitung vektorassoziierter Erkrankungen des Menschen in Deutschland unter Beriicksichtigung zoonotischer Aspekte. Bundesgesundheitsbl. - Gesundheitsforsch. - Gesundheitsschutz 44, 116-136 (2001). Foppa, I. M., Krause, P. J., Spielman, A., Goethert, H., Gern, L., Brand, B., Telford III, S. R.: Entomologic and serologic evidence of zoonotic transmission of Babesia microti, eastern Switzerland. Emerg. Infect. Dis. 8, 722-726 (2002). Gelfand, J.A.: Babesia. In: Mandell, G.L., Douglas, R. G., Bennett, J. E., Dolin, R.: Mandell, Douglas, and Bennett's Principles and Practice of infectious diseases. 5th Ed. Churchill Livingstone, New York, pp. 2899-2902 (2000). Gerber, M.A., Shapiro, E.D., Krause, P.J., Cable, R. G., Badon, S. J., Ryan, R. W.: The risk of acquiring Lyme disease or babesiosis from a blood transfusion. J. Infect. Dis. 170, 231-234 (1994). Gorenflot, A., Marjolet, M., Hostis, L., Coutarmanac'h, A., Marchand, A. : Existance probable en France de Babesioses humaines asymptomatique. In: Abstracts of the 3rd International Conference on Malaria and Babesiosis. Annecy, France: Fondation M. Merieux, abstract p. 134 (1987). Gorenflot, A., Moubri, K., Precigout, G., Carcy, B., Schetters, T. P.: Human babesiosis. Ann. Trop. Med. Parasitol. 92, 489-501 (1998). Granstr6m, M.: Tick-borne zoonoses in Europe. Clin. Microbiol. Infect. 3, 156-167 (1997). Gray, J., von Stedingk, L. V., Giirtelschmid, M., Granstr6m, M. Transmission studies of Babesia microti in Ixodes ricinus ticks and gerbils. J. Clin. Microbiol. 40., 1259-1263 (2002). Herwaldt, B. L., Persing, D. H., Precigout, E. A., Goff, W.L., Mathiesen, D.A., Taylor, P.W., Eberhard, M. L., Gorenflot, A. F.: A fatal case of babesiosis in Missouri: identification of another piroplasm that infects humans. Ann. Intern. Med. 124, 643-650 (1996).

102

K.-P. Hunfeld, V. Brade

Herwaldt, B. L., Caccio, S., Gherlinzoni, I., Asp6ck, H. Slemenda, S., Piccaluga, P. P., Martinelli, G., Edelhofer, R., Hollenstein, U., Poletti, G., Pampiglione, S., L6schenberger, K., Tura, S., Pieniazek, N. J.: Molecular characterization of a non-Babesia divergens organism causing zoonotic babesiosis in Europe. Emerg. Infect. Dis. 9, 942-948 (2003). Homer, M.J., Aguilar-Delfin, I., Telford III, S.R., Krause, P. J., Persing, D. H.: Babesiosis. Clin. Microbiol. Rev. 13, 451-469 (2000). Hunfeld, K.-P., Allwinn, R., Peters, S., Kraiczy, P., Brade, V.: Serologic evidence for tick-borne pathogens other than B. burgdorferi (TOBB) in Lyme borreliosis patients from midwestem Germany. Wien. Klin. Wochenschr. 24, 901-908 (1998a). Hunfeld, K.-P., Allwinn, R., Albert, S., Brade, V., Doerr, H.W.: Serologically proven double infection with tick-borne encephalitis virus (TBEV) and Borrelia burgdorferi. J. Lab. Med. 22, 409-413 (1998b). Hunfeld, K.-P., Brade, V.: Prevalence of antibodies against the human granulocytic ehrlichiosis agent in Lyme borreliosis patients from midwestern Germany. Eur. J. Clin. Microbiol. Infect Dis. 18, 221-224 (1999). Hunfeld K.-P., Cinatl, J., Tenter, A.M., Brade, V.: Granulocytic Ehrlichia, Babesia, and spotted fever Rickettsia: Not yet widely known tick-borne pathogens of considerable concern for humans in Europe. Biotest Bulletin 6 : 321 - 344 (2002a). Hunfeld K.-P., Lambert, A., Kampen, H., Albert, S., Epe, Ch., Brade, V., Tenter., A.: Seroprevalence of Babesia infections in humans exposed to ticks in midwestern Germany. J. Clin. Microbiol. 40, 2431-2436 (2002b). Huwer, M., Schwarzmaier, A., Hamel, H. D., Will, R.: The occurrence of B. divergens in the Freiburg i. Br. District and piroplasmosis prevention trials in cattle. Berl. Muench. Tier~irztl. Wochenschr 107, 901-908 (1994). Johansson, K. E., Pettersson, B., Uhlen, M., Gunnarsson, A., Malmqvist, M., Olesson, E.: Identification of granulocytic ehrlichiosis in Swedish dogs and horses by direct sequencing of PCR products from 16S rRNA gene. Res. Vet. Sci. 58, 109-112 (1995). Kampen, H., Maltezos, E., Pagonaki, M., Hunfeld, K.-P., Maier, W. A., Seitz, H. W.: Individual cases of autochthonous malaria in Evros Province, northern Greece: serological aspects. Parasitol. Res. 88, 261266 (2002). Kjemtrup, A. M., Conrad, P. A.: Human babesiosis: an emerging tick-borne disease. Int. J. Parasitol. 30, 1323 - 1337 (2000). Krause, P.J., Telford III, S.R.: Babesiosis. In: Gilles, H. M.: Protozoal diseases. Arnold, London, pp. 236248 (1999). Krause, P. J., Lepore, T., Sikand, V. K., Gadbaw, J. Jr., Burke, G., Telford III, S. R., Brassard, P., Pearl, D., Azlanzadeh, J., Christianson, D., McGrath, D., Spielman, A.: Atovaquone and azithromycin for the

treatment of babesiosis. N. Engl. J. Med. 343, 1454-1458 (2000). Krause, P. J., Telford III, S. R., Pollack, R. J., Ryan, R., Brassard, P., Zemel, L., Spielman, A.: Babesiosis: an underdiagnosed disease of children. Pediatrics 89, 1045-1048 (1992). Krause, P. J., Telford III, S. R., Ryan, R., Conrad, P. A., Wilson, M., Thomford, J. W., Spielman, A.: Diagnosis of babesiosis: evaluation of a serologic test for the detection of Babesia microti antibody. J. Infect. Dis. 169, 923-926 (1994). Krause, P. J., Telford III, S. R., Spielman, A., Sikand, V., Ryan, R., Christianson, D., Burke, G., Brassard, P., Pollack, R., Peck, J., Persing, D. H.: Concurrent Lyme disease and babesiosis: evidence for increased severity and duration of illness. J. Am. Med. Assoc. 275, 1657-1660 (1996). Levine, N. D.: The protozoan phylum Apicomplexa, vol. 2. CRC Press, Boca Raton (1988). Mehlhorn, H., Raether, W., Schein, E., Weber, M., Uphoff, M.: Licht- und elektronenmikroskopische Untersuchung der intraerythrozyt~iren Stadien yon Babesia microti. Dtsch. Tier~irztl. Wochenschr. 93, 400-405 (1986). Metz, W.: Einheimische und importierte zeckeniibertragene Infektionen bei Hunden in Deutschland. Inauguraldissertation, Tier~irztliche Hochschule, Hannover, p. 149 (1999). Mylonakis, E.: When to suspect and how to monitor babesiosis. Am. Fam. Phys. 63, 1969-1974 (2001). Nilsson, K., Lindquist, O., Liu, A. J., Jaenson, T. G. T., Friman, G., Pahlson, C.: Rickettsia helvetica in Ixodes ricinus ticks in Sweden. J. Clin. Microbiol. 37, 400403 (1999). Popovsky, M.A. Transfusion-transmitted babesiosis. Transfusion 31,296-298 (1991). Ricketts, H.T.: Some aspects of Rocky Mountain spotted fever as shown by recent investigations. Med. Rec. 16, 843-855 (1909). Sharan, K. P., Krause, P. J.: Babesiosis. In: B. A. Cunha: Tick-borne infectious diseases: diagnosis and management. Marcel Decker Inc., New York, pp. 111-120 (2000). Scheibe, K., Hahn, M., Oschmann, P.: Zecken6kologie and Epidemiologie. In: Oschmann, P. und Kraiczy, P.: Lyme borreliosis and FSME. Uni-Med Verlag, Bremen, pp. 2 6 - 3 0 (1998). Schouls, L. M., Van de Pol, I., Rijpkema, S. G. T., Schot, C. S.: Detection and identification of Ehrlichia, Borrelia burgdorferi s. l., and Bartonella spp. in Dutch I. ricinus ticks. J. Clin. Microbiol. 37, 2215-2222 (1999). Skotarczak, B., Cichocka, A.: Isolation and amplification by polymerase chain reaction DNA of Babesia microti and Babesia divergens in ticks in Poland. Ann. Agric. Environ. Med. 8, 187-189 (2001). Smith, T., Kilbourne, E. L.: Investigation into the nature, causation, and prevention of southern cattle fever. US Dept. Agr. Bur. Anim. Indust. Bull. 1 (1893).

Babesiosis in humans Uhnoo, I., Cars, O., Christensson, D., Nystroem-Rosander, C.: First documented case of human babesiosis in Sweden. Scand. J. Infect. Dis. 24, 541-547 (1992). Walter, G.: Zur Obertragung und zum Parasit/imieverlauf von Babesia microti (Stature 'Hannover') bei R6telmaus (Clethrionomys glareolus) und Erdmaus (Microtus agrestis). Acta Trop. 41,259-264 (1984). Weis, E.: History of rickettsiology. In: Walker, D.H.: Biology of rickettsial diseases. CRC Press, Boca Raton, pp. 15-32 (1988).

103

White, D. J., Talarico, J., Chang, H. G., Birkhead, G. S., Heimberger, T., Morse, D. L.: Human babesiosis in New York state: Review of 139 hospitalized cases and analysis of prognostic factors. Arch. Intern. Med. 158, 2149-2154 (1998). Wilske, B., Z611er, L., Brade, V., Eiffert, H., Goebel, U. B., Stanek, G., Pfister, H. W.: Lyme Borreliose. In. Mauch, H., Liittiken R. (Eds.): Qualit/itsstandards in der mikrobiologisch-infektiologischen Diagnostik. Urban & Fischer Verlag, Miinchen, Germany (2000).