Epidemiology and Control of Human Granulocytic Anaplasmosis: A Systematic Review

Abstract

Granulocytic anaplasmosis caused by Anaplasma phagocytophilum is an emerging tick-borne zoonosis worldwide. The obligate intracellular pathogen is transmitted by Ixodes ticks and infects neutrophils in humans and animals, resulting in clinical symptoms ranging from asymptomatic seroconversion to mild, severe, or fatal disease. Since the initial description of human granulocytic anaplasmosis (HGA) in the United States in 1990, HGA has been increasingly recognized in America, Europe, and Asia. This review describes the epidemiology, diagnosis, and treatment of HGA and provides background information on the potential vectors and reservoirs of A. phagocytophilum.

Introduction

T he obligate intracellular pathogen Anaplasma phagocytophilum, which includes the former Ehrlichia phagocytophila, Ehrlichia equi, and the human granulocytic ehrlichiosis agent, is an emerging tick-borne pathogen in humans and animals worldwide. The organism was first reported in sheep from Scotland. The disease in domestic ruminants caused by A. phagocytophilum is also known as tick-borne fever and has been reported in sheep, cattle, goats, and deer. Equine granulocytic anaplasmosis was documented as early as 1968 in California. Infection of dogs was first identified in California in 1982. All these agents have been reclassified and recognized as a single species, A. phagocytophilum, based on molecular phylogenetic analysis (Dumler et al. 2001).

A. phagocytophilum is a small, gram-negative, obligate intracellular pathogen that primarily parasitizes the membrane-bound and cytoplasmic vacuoles of neutrophils (Scorpio et al. 2005). Infection of the neutrophils results into inflammatory lesions, which are most evident in liver lobules and lymph nodes (Johns et al. 2009).

A. phagocytophilum can be cultured in vitro in the human promyelocytic cell line HL-60 and in the I. scapularis tick-derived cell lines ISE6 and IDE8 (Woldehiwet et al. 2002, Zeman et al. 2002). The genome of A. phagocytophilum is about 1,471,282 bp with a G+C content of 41.63%. Although the functions of many genes are not completely known, some, including the large repertoire of outer membrane proteins and some of the virulence determinants, such as type IV secretion system proteins, may contribute to the survival of A. phagocytophilum in different environments (Dunning Hotopp et al. 2006).

Human granulocytic anaplasmosis (HGA) often presents as a nonspecific febrile illness, ranging from asymptomatic infection to fatal disease. Nonspecific symptoms include acute onset fever, headache, malaise, and myalgias, and the less common signs include nausea, abdominal pain, diarrhea, and cough. Hematologic disorders, including leukopenia, lymphopenia, and thrombocytopenia, are also observed. Most symptoms usually resolve within 30 days, even without treatment (Dumler et al. 2005, Dumler et al. 2007).

Leukopenia and lymphopenia in HGA patients can cause secondary, opportunistic infections with organisms such as Candida albicans (Garyu et al. 2005). Although the fatality rate is <1%, various complications, such as septic or toxic shock-like syndrome, respiratory insufficiency, rhabdomyolysis, pancarditis, acute renal failure, and hemorrhage, can occur several days or longer after onset (Hilton et al. 2008).

Epidemiology of HGA

Since the first case was reported in the United States in 1990, HGA has been found in both Europe and Asia. In the United States, about 5000 cases of HGA had been reported as of 2008 according to provisional cases of selected notifiable diseases reported to the Center for Disease Control (Atlanta, GA). The actual number of cases may be much higher, because many cases are neither reported nor even recognized because of the nonspecific clinical presentation of HGA. Most cases are reported in the Northeast and upper Midwest, especially in Rhode Island, Minnesota, Connecticut, New York, and Maryland. The average annual incidence nationwide was 1.4 cases per million people, with a seroprevalence ranging from 0.6% to 14.9% in endemic areas. The median age of patients was 51 years, and the highest age-specific incidence occurred among persons aged 60–69 years because of the high probability of exposure to ticks (Graf et al. 2008).

An investigation conducted in Connecticut showed a seroprevalence of 3.5% in blood donors, 4.2% of whom reported having been bitten by a tick (Leiby et al. 2002). The exposure of blood donors to A. phagocytophilum suggests a need to better understand the transfusion transmission potential of the bacterium.

The first European HGA case was reported in Slovenia in 1995 (Lotric-Furlan et al. 2006). Although only a small number of laboratory-confirmed HGA cases have been reported in European countries, the disease has been found in the Netherlands (van Dobbenburgh et al. 1999), Germany (Kowalski et al. 2006), France (van Dobbenburgh et al. 1999), Italy (Mastrandrea et al. 2006), Spain (Oteo et al. 2000), Sweden (Oteo et al. 2000), Norway (Kristiansen et al. 2001), Austria (Walder et al. 2006), Denmark, Poland, and Croatia (Misic-Majerus et al. 2006). The seroprevalence was 9% in Austria (Walder et al. 2003), 9.6%–19.8% in Poland (Stanczak and Grzeszczuk 2006), 1.4% in Spain (Blanco and Oteo 2002), 7.3% in Greece (Daniel et al. 2002), 5.7% in Italy (de la Fuente et al. 2005), 21% in Denmark (Skarphedinsson et al. 2001), and 1.4%–2.8% in Germany (Hunfeld and Brade 1999, Kowalski et al. 2006). The proportion of seropositive persons increases with age and is higher in humans with a history of having been bitten by a tick (Graf et al. 2008).

A. phagocytophilum infection may also occur in patients with other tick-borne diseases. In Italy, antibodies to A. phagocytophilum were detected in patients co-infected with Borrelia burgdorferi sensu lato (s.l.), the pathogen that causes Lyme borreliosis (LB) (Beltrame et al. 2006). The seroprevalence of antibodies for A. phagocytophilum and B. burgdorferi s.l. was 0.93% in Slovakia (Kalinova et al. 2009). In Portugal, 7.8% of LB patients and 0.4% of LB-seronegative patients had confirmed HGA, suggesting that LB patients are significantly more likely to come into contact with A. phagocytophilum (Santos et al. 2006). In the Czech Republic, 9.9% of patients hospitalized for suspected tick-borne encephalitis had IgG antibodies for A. phagocytophilum (Zeman et al. 2007). Coxiella burnetii has also been isolated from HGA patients (Santos et al. 2009).

HGA is also present in Asia, based on serological and molecular evidence. In China, the first HGA case was found in Anhui Province in 2006. A 50-year-old woman, bitten by a tick 12 days earlier, died of HGA. Subsequently, nine patients, including relatives, doctors, and nurses, were confirmed to have been infected with A. phagocytophilum, whose transmission occurs via the blood or respiratory secretions (Zhang et al. 2008). This is the first report of person-to-person transmission of HGA.

At least 557 people have been found to be infected with A. phagocytophilum in China, distributed across Hubei, Henan, Anhui, Shandong, Helongjiang, Inner Mongolia, Xingjiang, and Tianjin, and 18 of them have died in the past 5 years (Zhang et al. 2008). Antibodies for A. phagocytophilum were detected in 20% of the individuals at high risk for exposure to ticks and animals in central and southeastern China (Zhang et al. 2009).

In Korea, the seroprevalence of A. phagocytophilum was 1.8%, although no HGA cases had been described (Heo et al. 2002).

Hosts for A. phagocytophilum and Their Roles As Reservoirs

In addition to human beings, many domestic animals, such as dogs, cats, horses, sheep, goats, and cattle, can also become infected with A. phagocytophilum and show clinical symptoms. These animals can act as sentinels for human A. phagocytophilum infection and may become sources of infection.

A competent reservoir of A. phagocytophilum must be naturally exposed to A. phagocytophilum and susceptible to infection; if this is the case, the organism can transmit A. phagocytophilum to uninfected vectors under natural conditions. A. phagocytophilum is naturally maintained in a cycle between ticks and rodents.

In the eastern United States, white-footed mice (Peromyscus leucopus) are considered a prominent reservoir (Telford et al. 1996). Actually, white-footed mice are a poor reservoir for A. phagocytophilum, because infection in mice is transient, possibly resulting from host immunity (Levin and Fish 2000). This phenomenon suggests that nonperomyscus reservoirs may exist in the United States. Raccoons (Procyon lotor) and eastern gray squirrels (Sciurus carolinensis) meet the requirements of reservoir competence for A. phagocytophilum, and raccoons seem to play a more important role in the amplification of A. phagocytophilum infection in ticks than white-footed mice (Levin et al. 2002, Yabsley et al. 2008). White-footed mice are the most common host of A. phagocytophilum and B. burgdorferi s.l., but primary infection with one agent will inhibit transmission of another (Levin and Fish 2001).

White-tailed deer (Odocoilius virginianus) harbor a variant strain of A. phagocytophilum that is not associated with human disease and cannot establish an infection in mice (Massung et al. 2003, Massung et al. 2005). Therefore, white-tailed deer are not a reservoir for strains that cause human disease.

In the western United States, dusky-footed wood rats (Neotoma fuscipes) can be infected with several A. phagocytophilum strains, but they cannot transmit the pathogen to other animals via ticks (Nieto et al. 2010). Moreover, the A. phagocytophilum msp2 gene sequence in wood rats is different from that in humans, horses, or other nonrodent vertebrates (Barbet et al. 2006). Therefore, wood rats are not reservoir competent. Unlike in the eastern United States, where a single vector species and a dominant reservoir host species maintain a sylvatic cycle, in the western United States there are multiple hosts that maintain A. phagocytophilum, including gray squirrels (Sciurus griseus) and chipmunks (Tamias), and potentially more than one vector species transmits and maintains A. phagocytophilum.

A. phagocytophilum may be maintained in a parallel enzootic cycle between cottontail rabbits and the Ixodes dentatus tick on Nantucket Island. I. dentatus does not bite humans, but it may serve as the bridge between the rabbit cycle and the mouse cycle of A. phagocytophilum. Multiple cryptic cycles exist in nature, which may help A. phagocytophilum avoid local extinction (Goethert and Telford 2003).

Serologically positive lizards and snakes were also found in the United States, but they were difficult to infect with A. phagocytophilum experimentally, and Ixodes pacificus that fed on an infected lizard did not transmit the pathogen, suggesting that the reptiles are not reservoir competent (Nieto et al. 2009).

Gray foxes (Urocyon cinereoargenteus), whose geographic distribution coincides with that of granulocytic anaplasmosis in humans and domestic animals in North America, are common in northwestern California, and the seroprevalence among gray foxes is similar to that of domestic dogs. Therefore, gray foxes are good sentinel animals for A. phagocytophilum infection (Gabriel et al. 2009).

The low prevalence of A. phagocytophilum in ticks from black bears (Ursus americanus) suggests that black bears may not be important hosts of A. phagocytophilum (Yabsley et al. 2009). Opossums (Didelphis virginiana), shrews (Blarina and Sorex), and striped skunks (Mephitis mephitis) can become naturally infected with A. phagocytophilum, but they fail to transmit the agent to uninfected vectors under natural conditions (Levin et al. 2002).

In Europe, wood mice (Apodemus sylvaticus) and bank voles (Clethrionomys glareolus) are the most commonly reported reservoirs (Bown et al. 2003). A. phagocytophilum can be maintained in a natural cycle between woodland rodents and Ixodes trianguliceps in United Kingdom, where bank voles are significantly more likely to be infected with A. phagocytophilum than wood mice. It may be that greater numbers of nymphs are carried by bank voles than by wood mice, or the behavioral characteristics of bank voles may make them more likely to encounter ticks (Bown et al. 2003).

Wild cervids, including roe deer and red deer, are important reservoirs of A. phagocytophilum in Europe. Experimental exposure to A. phagocytophilum in red deer has shown that a subclinical infection can develop in these animals (Stuen et al. 2001). Wild ruminants are naturally exposed to A. phagocytophilum, which is the most widespread tick-borne infection in animals in Europe (Stuen 2007). In Norway, A. phagocytophilum seroprevalences of 43%, 55%, and 96% have been found in moose, red deer, and roe deer, respectively; these are the highest rates in Europe (Stuen et al. 2002). Wild cervids clearly play an important role in maintaining the natural cycle of A. phagocytophilum in Europe.

Studies of the prevalence of A. phagocytophilum infection in wild boars indicate that A. phagocytophilum can naturally establish a stable cycle in wild boars, and the groESL gene of A. phagocytophilum from wild boars in Slovenia is identical to that in other European countries. This provides molecular evidence of a zoonotic cycle of A. phagocytophilum between Ixodes ricinus ticks, wild boars, and humans in Europe (Haydon et al. 2002, Strasek Smrdel et al. 2009).

The presence of A. phagocytophilum was shown in 1.3% of 76 common shrews (Sorex araneus) from northwestern England (Bray et al. 2007). Recent xenodiagnostic assays have confirmed A. phagocytophilum infections in eastern gray squirrels (S. carolinensis), raccoons, and eastern cottontail rabbits (Sciurus floridanus), indicating that these mammals are competent reservoirs for this pathogen in the New England region. Although A. phagocytophilum strains from European roe deer (Capreolus capreolus) may not be involved in human disease, roe deer may serve as a wildlife reservoir for transmission to domestic and wild animals in southern Spain (de la Fuente et al. 2008).

In Asia, Apodemus agrarius is considered the most important reservoir of A. phagocytophilum. Three A. phagocytophilum strains have been isolated from A. agrarius and Tamias triton rodents in China, suggesting that these wild animals may act as competent reservoirs of A. phagocytophilum (Zhan et al. 2010). The average prevalence of A. phagocytophilum in A. agrarius was about 11.4% in China (Cao et al. 2006, Zhan et al. 2009) and 4.5% in Korea (Chae et al. 2008). Feral raccoons might be infected with A. phagocytophilum in Japan, according to serological tests (Inokuma et al. 2007). These data suggest that wild animals may contribute to the maintenance of a natural cycle of A. phagocytophilum in Asia, but the natural reservoir of the bacterium has not been experimentally confirmed.

Ixodes Tick Vectors

In the United States, I. scapularis, which lives in humid forests, is the principal vector of A. phagocytophilum. In the North, adult ticks are active from March to June and from October to December; nymphs are active in spring and summer; larvae are active from June to October (Keirans et al. 1996). In the South, all life stages of the tick are active from November to May (Parola et al. 2005). I. pacificus, which lives in shrub forests and deserts, is the primary vector in the Pacific coast states (Parola et al. 2005). I. dentatus is another vector of A. phagocytophilum in cottontail rabbits and wood rats in the United States, and it can also be transported by birds, contributing to the introduction of the pathogen to new areas (Goethert and Telford 2003). A molecular survey of A. phagocytophilum showed that the mean prevalence in the United States is 15.0% in I. scapularis, 3.8% in I. pacificus, and 4.7% in I. dentatus.

Because of its capacity for transovarial transmission, Dermacentor albipictus may be another vector of A. phagocytophilum variants in white-tailed deer (Baldridge et al. 2009).

In Europe, I. ricinus, which inhabits forests and pastures, is the main vector of A. phagocytophilum. All life stages of the tick are usually active from April to June. A. phagocytophilum has been detected in I. ricinus throughout Europe. For example, the prevalence of A. phagocytophilum in I. ricinus was 5.1% in Austria (Polin et al. 2004), 8.7% in Poland (Grzeszczuk et al. 2004), 1.4% in Switzerland (Liz 2002), and 4% in Portugal (Santos et al. 2004). However, the dynamics of transmission to mammals have not been completely elucidated. I. trianguliceps is also a competent vector, and it can maintain A. phagocytophilum in a natural cycle with woodland rodents in Great Britain (Bown et al. 2008).

In Asia, I. persulcatus is considered the primary vector of A. phagocytophilum. The prevalence of A. phagocytophilum in I. persulcatus was 4.6% in China (Cao et al. 2003, 2006) and 9.6% in Japan (Ohashi et al. 2005). In Japan, I. persulcatus and I. ovatus ticks have been shown to be naturally infected with A. phagocytophilum and are able to transmit A. phagocytophilum. Haemaphysalis megaspinosa has been shown to harbor A. phagocytophilum (12.5%), but its capacity as a vector should be further validated (Yoshimoto et al. 2010).

Diagnosis

Patients with HGA usually present nonspecific clinical symptoms. Although leukopenia and thrombocytopenia occur during the early stage, these signs often disappear by the end of the second week. Therefore, laboratory confirmation is necessary.

During the early stage of the infection, the quickest diagnostic method is microscopic examination of peripheral blood smears. The intravacuolar bacterial inclusions (morulae), which are indicative of infection, can be found in neutrophils. The accuracy of this method is 25%–75% during the first week and declines thereafter (Aguero-Rosenfeld 2002).

Polymerase chain reaction (PCR) is currently the most sensitive method to detect A. phagocytophilum during the early stage of the infection. After the first week, the bacteremia rapidly wanes and the sensitivity of PCR decreases. The sensitivity of PCR is 67%–90% (Dumler and Brouqui 2004). Multiplex real-time PCR can also be used as rapid diagnostic methods, with the advantage of providing concurrent detection of two or more ehrlichiosis and anaplasmosis agents and avoidance of gel analysis (Courtney et al. 2004).

Isolation of A. phagocytophilum from a patient is a definitive diagnosis of infection. A. phagocytophilum is usually cultivated in the human promyelocytic leukemia cell line HL-60 and tick embryo cell lines. The bacteria develop within vacuoles and form morulae in the cytoplasm of infected cells at 5–14 days after inoculation.

The indirect immunofluorescent antibody assay is the most sensitive method to detect antibodies against A. phagocytophilum, and it is commonly used to confirm HGA. The sensitivity of detection is higher during the 2–4 weeks following disease onset, compared with PCR, blood smear microscopy, or cell culture (Bakken and Dumler 2006).

Treatment

The current recommended therapy for HGA is administration of doxycycline or tetracycline for 5–14 days. The recommended dosage of doxycycline is 100 mg for adults and 2.2 mg/kg for children aged 8 years or older, given orally every 12 h. Tetracycline should be administered orally every 6 h at a dosage of 500 mg for adults and 25–50 mg/kg/day for children aged 8 years or older (Dumler et al. 2007). Treatment with doxycycline and tetracycline can effectively improve overall wellbeing and fever within 24–48 h. Rifampin is recommended only for patients with a history of allergy to tetracycline antibiotics, children under 8 years of age, or women who are pregnant. The dosage is 300 mg orally twice daily for adults or 10 mg/kg for children (maximum of 300 mg/dose). Treatment should continue until the patient is afebrile for 3 days (Krause et al. 2003, Bakken and Dumler 2006).

Prevention

Because A. phagocytophilum can be maintained naturally in a rodent–tick cycle and a large number of vertebrate animals are susceptible to it worldwide, it would be very difficult to eliminate this pathogen from the environment. Because the main route of transmission of A. phagocytophilum is via tick bites, the most effective method for prevention of HGA is to avoid exposure to ticks. Wearing protective clothing and applying repellent sprays can reduce the risk of tick attachment. N,N-Diethyl-meta-toluamide (DEET), permethrin, and some natural compounds, such as citrodiol and p-menthane-3,8-diol, are considered safe and effective for preventing tick attachment (Piesman and Eisen 2008).

Nontick transmission of HGA, including direct exposure to deer blood, transfusion, and transplacental and person-to-person transmission, has also been reported (Zhang et al. 2008). Therefore, direct contact with blood and excretions from patients and animals infected with A. phagocytophilum should be avoided.

For HGA patients, it is critical to obtain an early diagnosis and to initiate antibiotic therapy as soon as possible. This is especially important in areas where Ixodes ticks are endemic. Doxycycline is the best treatment, but it cannot be prescribed to pregnant women, young children, or individuals who have a history of allergy to doxycycline or tetracycline. For those unable to take doxycycline or tetracycline, rifampin is an alternative (Dumler et al. 2007).

Surveillance of A. phagocytophilum infection in animals and tick vectors should continuously identify endemic areas and predict epidemiological trends. This will contribute to the prevention and control of HGA throughout the world.

Footnotes

Acknowledgments

This study is supported in part by grants from the National Key Technology R&D Program in China (2010BAD04B01) and the National Natural Science Foundation of China (30972178).

Disclosure Statement

No competing financial interests exist.

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