Diagnosis and management of human babesiosis.

Babesiosis is a protozoan parasitic infection affecting humans and animals. These infections are commonly transmitted by various species of Ixodes ticks depending upon the geographical location. They can also be transmitted by packed cell transfusion and transplacental route from mother to child. Various species have been reported to cause human infections, of which Babesia microti is the most common species reported globally. Usually, Babesia infections are asymptomatic or mild, but can be severe/life-threatening in immunosuppressed or splenectomized individuals. A high index of clinical suspicion in residents of endemic areas or individuals who had a recent travel history to such areas, with laboratory confirmation can guide an early institution of appropriate antimicrobial therapy, thereby preventing complications and death.

INTRODUCTION

Babesiosis is a zoonotic disease of humans caused by the hemoprotozoan piroplasm parasite “Babesia.” The primary reservoir hosts are usually the rodents, primarily the white-footed mice. Several wild and domestic animals serve as incompetent reservoir hosts depending on the species involved and help in the maintenance of the transmitting vector, Ixodes spp. Humans act as accidental host and acquire infection by the bite of Ixodes ticks. Human to human transmission can occur by transfusion of packed red cells or rarely by transplacental route. The disease has been reported both in immunocompetent and immunocompromised individuals. Depending on the immune status and risk factors, the disease severity can vary from asymptomatic or mild febrile illness to life-threatening complications. The risk factors associated with severe babesiosis are old age, asplenia/hyposplenism, HIV-infected individuals, malignancy, and patients on immunosuppressive agents particularly rituximab and on tumor necrosis factor-alpha inhibitors like infliximab and etanercept.[12] Earlier, Babesia spp. causing human infections (Babesia spp. sensu stricto) were distinguished from closely related Theileria spp. causing animal infections by the ability to transmit transovarially in adult ticks and inability to invade host lymphocytes. Babesia spp. sensu stricto was characterized by the formation of trophozoites >3 μm size and existing as pairs of merozoites in red blood cell (RBC). Later, identification of small Babesia spp. (<3 μm size) which neither invades host lymphocytes nor transmitted transovarially in adult ticks but characteristically undergo double binary fission producing 4 merozoites, made the classification obsolete.[3] Currently, the piroplasms causing human infections are classified under 5 clades phylogenetically. The first clade contains Babesia microti and B. microti-like (previously KO-1). The second clade constitutes the B. duncani (previously WA-1) and B. duncani-type (previously CA1-4) organisms. The third clade constitutes the Theileria spp. The fourth clade constitutes the Babesia divergens, B. divergens-like (previously MO-1), and Babesia venatorum (previously EU-1). The fifth clade constitutes the Babesia bovis and Babesia bigemina. The phylogenetic fourth and fifth clades are related to the large Babesia group that is, Babesia spp. sensu stricto. Although the fourth clade is related to the large Babesia group, the trophozoites formed inside RBC's are < 3 μm size.[4] Most human Babesia infections are seen in the United States and Europe, and few in China, Egypt, Mozambique, South Africa, Japan, Korea, and Taiwan. In the United States, B. microti, B. duncani, and B. duncani-type human infections are common with B. microti being the most prevalent species. In contrast, B. divergens and B. venatorum are the prevalent species causing human infections in Europe. Recently, B. venatorum was found to be endemic in China, in addition to sporadic B. microti and B. divergens infections.[5678] Febrile cases seen in residents of endemic areas, recent travel, or blood transfusion should raise clinical suspicion of babesiosis. Early clinical diagnosis with laboratory confirmation is a key to prevent mortality commonly seen in splenectomized or immunocompromised individuals. Here, we discuss the clinical and laboratory features in human babesiosis infections.

CLINICAL MANIFESTATIONS

Babesiosis presents as a broad spectrum of illness ranging from asymptomatic to life-threatening illness in humans. The severity of the illness depends primarily on the presence of risk factors, the species involved, and the presence of associated tick-borne illnesses such as ehrlichiosis, anaplasmosis, and borreliosis.[910] The incubation period varies depending on the route of transmission usually lasting 1–4 weeks following a tick bite, and slightly longer (up to 6 weeks) following transfusion of contaminated blood products. Most infected cases remain asymptomatic while few, particularly with associated risk factors develop symptomatic infection. Approximately, 50% of children and 25% of adults remain asymptomatic.[1112] Postincubation period, infected individuals develop fatigue, myalgia, and malaise followed by persistent or intermittent pattern of fever. Fever has been a consistent symptom in symptomatic babesiosis ranging from mild to high-grade reaching up to 40.9°C. Other commonly reported symptoms were chills, sweats, headache, anorexia, vomiting, diarrhea, abdominal pain, joint pain, sore throat, cough, breathing difficulty, redness of eyes, dark-colored urine, depression, and emotional lability. The symptoms reported were highly nonspecific which could pertain to many other febrile infective conditions. Commonly observed signs were fever, pharyngeal erythema, hepatomegaly, splenomegaly, jaundice, and retinal hemorrhages. Rash was observed in cases with co-existing Lyme borreliosis, characteristically migrating (erythema chronicum migrans).[10] Blood parameters typically reveal a scenario of hemolytic anemia, characterized by decreased hemoglobin, haptoglobin, and hematocrit with increased bilirubin, reticulocyte count, lactate dehydrogenase, urobilinogen, and urine hemoglobin. The laboratory picture of hemolytic anemia with raised liver enzymes (aspartate and alanine transaminase, alkaline phosphatase), thrombocytopenia and variable leukocyte count in a clinically suspected case can differentiate babesiosis from similar febrile infective syndromes.[1] Neutropenia is observed in neonatal babesiosis and also should raise suspicion of co-existing granulocytic anaplasmosis.[13] Bone marrow examination may reveal increased erythropoiesis and occasionally hemophagocytosis. Complications can develop in approximately 5–10% of cases whereas it can range to 20% in cases of immunosuppression and transfusion-transmitted babesiosis (TTB). The common complications reported were acute respiratory failure, congestive cardiac failure, renal failure, disseminated intravascular coagulation, and splenic rupture despite adequate treatment. High parasitemia (>10%) and severe anemia (<10 g/dl) were found to predict the development of complications and subsequent death.[14] Symptoms usually subside within few days on adequate treatment, but can last weeks to months in certain cases. Persistent infection or relapses have been reported commonly in immunosuppressed individuals.[15] The fatal outcome has been reported in all Babesia spp. except B. venatorum.[67] In particular, cases infected with B. divergens and B. duncani species have been found to manifest as severe disease and subsequent fatal outcome.[1617]

LABORATORY DIAGNOSIS

The clinical manifestations and the signs observed in cases of babesiosis are not pathognomonic and hence for definitive diagnosis and appropriate treatment, microbiological identification is necessary. Various methods have been employed for identification of this parasitic infection that is, microscopic detection, culture, serological tests, and molecular techniques.

Microscopy

Examination of peripheral thick and thin blood smears has been the standard technique used in the diagnosis of babesiosis in humans. Romanowsky stains like Giemsa and Wright stains have been widely utilized for staining of blood films for microbiological examination.[18] Ring forms are observed which resembles Plasmodium falciparum trophozoites. These ring forms can be round, oval, pyriform, or amoeboid shaped existing in singles, pairs, or rarely in tetrads characteristically termed as the “Maltese-cross appearance.” The differentiating features of Babesia from P. falciparum are the absence of hemozoin pigments in infected RBC, absence of schizonts and gametocytes, presence of extracellular forms and typical “Maltese-cross pattern.”[1920] The size of the infected RBC's remains normal, and the cytoplasm of the ring form remains clear, particularly in large Babesia infections due to the presence of vacuole whereas remains pink like the rest of RBC cytoplasm in Plasmodium infections. Parasitemia in babesiosis can range from 1% to around 80% in severe infections. The percentage parasitemia should be calculated in all positive cases. In high parasitemia cases, thin blood film is used for the determination of percentage parasitemia where the number of infected RBC's per 100 RBC counted is taken. In low parasitemia or thick smear evaluation, evaluating number of parasites per 200 white blood cells (WBC) and the WBC count in the patient determines the parasite density. Babesia infections can be easily missed in thin smear evaluation if 300 fields or multiple smears are not examined in cases with low parasitemia.[21] Similarly, automated blood analyzers can also lead to false-negative results due to the low volume of blood and/or fields being analyzed.[22] Species determination is of epidemiologic and to some extent of therapeutic importance since B. divergens/B. bovis infections are usually severe requiring exchange transfusion. In the microscopic examination, large Babesia (trophozoites >3 μm) typical of the fifth clade (B. bovis/B. bigemina) can be differentiated from other species (small Babesia – trophozoites <3 μm). The maltese-cross pattern is a characteristic of B. microti/B. duncani, but rarely has also been reported in infections with other Babesia spp.[23] Microscopically, it is impossible to differentiate B. microti from B. duncani infections. B. divergens and B. bovis infections are characterized by paired merozoites and occasionally can be differentiated by the diverging angle of around 180° between the pyriform/oval merozoites characteristic of B. divergens.[24] Although these features theoretically seem to make a distinction, in practical situations, species differentiation can be difficult using microscopic examination. Quantitative buffy coat (QBC) examination used widely for diagnosis of malaria can also detect the infected RBC's but poses difficulty in differentiating Plasmodium and Babesia. Hence, Giemsa-stained blood smears are preferred over QBC examination. Although electron microscopy has been utilized to detect and study Babesia from human blood, it is not a useful tool for routine diagnosis of human babesiosis.

Culture

Culture techniques had been widely used in the diagnosis of Babesia infections in animals. This method has not been used for the diagnosis of human infections since it is time-consuming, laborious, and gives highly variable results. Yet culture methodology has helped in identification of asymptomatic/low-parasitemic individuals, defining phylogenetic relationships, production of antigens for in vitro diagnostic and vaccine preparation purposes, study host-parasite interactions, and antimicrobial susceptibility testing. Cultivation of these parasites can be performed in artificial media or by animal inoculation. Commonly employed animals are hamsters, severe combined immunodeficiency (SCID) mice, gerbils, and splenectomized calves. SCID mice whose circulating RBC's are replaced with human erythrocytes (Hu-RBC SCID mice) have been used to detect B. microti-like human infections.[25] These animals are inoculated intraperitoneally; the suspected blood sample and after 2–4 weeks, blood samples are collected from the tail vein for detection of the parasites. B. duncani has been found to be lethal in hamsters and mice (lethality in < 10 days) and B. divergens grow well in gerbils.[20] Xenodiagnoses that is, detection of Babesia spp. from ticks, either by microscopy, culture or molecular techniques has been employed for supportive epidemiological evidence in the diagnosis of babesiosis.[26] Numerous artificial media and culture techniques used in the diagnosis of malaria have been utilized in the cultivation of Babesia spp. from animals that can also be utilized in the diagnosis of human infections. The media used is buffered (HEPES/TES/RPMI-1640) containing serum and added antibiotics. The choice of the buffer and the serum utilized differs based on the Babesia spp. suspected. Microaerophilous stationary phase culture technique has been a method used to detect Babesia infection in asymptomatic/low-parasitemic individuals or animals. The blood sample from suspected cases/animals is defibrinated and subsequently mixed with the buffered medium and serum depending on the species suspected (based on the prevalent species). This mixture is transferred to culture vessels and incubated in 5% CO2 atmosphere at 37°C. At 2–4 weeks incubation, the parasitemia is amplified and can be detected by microscopic examination. Every 48–72 h, the culture is diluted 3–25-fold with uninfected RBC's of the same species. This technique thus maintains a continuous culture medium that serves antigens for diagnostic and vaccine preparation purposes.[27]

Serological tests

Serological tests have been used widely in endemic areas to support or confirm the diagnosis of human babesiosis. These techniques are particularly useful in screening blood donors for Babesia infections since asymptomatic/low-parasitemic individuals can be easily missed in the blood smear examination. The techniques available for detection of specific antibodies are indirect immunofluorescence assay (IFA), enzyme linked immune-sorbent assay (ELISA), and immunoblot. Among these techniques, IFA detecting specific IgG antibodies has been extensively utilized as the serological test of choice in most endemic areas. Centers for Disease Control (CDC) has proposed and made available a B. microti - IgG antibody detection kit (IFA) for diagnostic purposes. Individuals with titers >1:64 were termed seropositive. Patients with acute B. microti infection had antibody titers >1:1024, which fell to 1:64 in 8–12 months. A 4-fold rise in titer after 2–4 weeks can confirm an infection in endemic areas when a single low titer value (>1:64) is obtained.[28] The main drawback of IgG antibody detection is its inability to differentiate recent from past infection. IgM antibody detection can detect and differentiate recent from the past infection, but has the disadvantage of false-positive results. IgG antibody cut-off titers can vary for different regions and species causing infections. Hence, the cut-off titers need to be calculated bases of the seroprevalence rate and the predominant circulating species. Cross-reactions have been observed with cases of malaria, but samples seropositive for B. microti do not cross-react with other Babesia spp. antigens. IgG antibody detection is not useful in the diagnosis of B. divergens infection since the course of illness is rapid and fulminant. Several immunodominant antigenic peptides were identified from Babesia merozoite surface proteins (BMN1-17 and MN-10) and have been employed in ELISA for detection of antibodies in blood donors.[2930] A better antigenic peptide to surface peptide that is, BmSA1 – a secretory antigen has been found to be highly sensitive and specific in detecting seropositive individuals.[31]

Nucleic acid amplification and sequencing

Molecular methods have emerged as the diagnostic method of choice for most infectious diseases including babesiosis. These methods have been used to confirm infection in seropositive individuals and to monitor treatment response. The most commonly employed target in the detection of Babesia spp. is 18S rRNA. Numerous conventional and real-time polymerase chain reaction (PCR) methods have been reported. A recent study utilizing real-time PCR technique reported a high sensitivity of detecting 20 genome copies/μl of blood that is, approximately 0.0001% parasitemia.[32] The detection and confirmation of novel strains of Babesia spp. causing human infections have also been possible due to the genome sequencing methods utilized for phylogenetic analysis. B. duncani was discovered from a patient in Washington, who presented with malaria-like illness and microscopic examination revealing morphology similar to B. microti. This strain differed from B. microti in growing continuously in stationary cultures, being lethal to hamsters, antigenic cross-reactivity, and restriction fragment length polymorphism patterns. The subsequent molecular phylogenetic analysis revealed that the strain was closely related to Babesia gibsoni, a canine pathogen. Hence, B. duncani was previously labeled as WA-1 strain.[233] Similar was the discovery of “B. duncani-type” (CA 1–4) infections detected in four cases of California, where the strain resembled B. duncani (WA-1) yet the phylogenetic study revealed it to be distinct from B. duncani (WA-1) and B. gibsoni.[34] While B. microti, WA-1, and WA-like were the species identified causing human infections in the Unites States, a novel strain from Missouri not reacting with any of these antigens in IFA but phylogenetically closely related to B. divergens was reported. This novel strain is now referred as “B. divergens-like” (previously MO-1).[3536] Similarly, “B. microti-like” strains were reported from Japan and Taiwan, and another novel strain reported from Korea, labeled as KO-1 strain.[37] Determination of sequence variations in amplified targets of internal transcribed spacers of nuclear rRNA can reliably differentiate B. microti from closely related species B. duncani.[38]

TREATMENT AND PREVENTION

Based on the recommendations of Infectious Diseases Society of America, asymptomatic babesiosis (with parasitemia persisting <3 months) and suspected cases without laboratory confirmation do not require treatment.[39] The therapeutic strategies depend on the severity of the infection, infecting species, and the immune status of the individual. For mild infections, a combination of oral atovaquone with azithromycin is recommended for a period of 7–10 days and has been found to be as efficacious as clindamycin-quinine combination used in the treatment of severe infections.[4041] In immunocompromised individuals, a higher dose of azithromycin (600–1000 mg/day) has been recommended for cure.[42] In case of severe infections (hemoglobin <10 g/dl, parasitemia >10%, B. divergens/B. bovis/B. duncani infections), partial or complete RBC exchange transfusion with oral/intravenous clindamycin plus oral quinine/intravenous quinidine is recommended.[3943] Persistent or relapsing infections are common in immunosuppressed individuals and hence prolonged treatment for 6 weeks with 2 weeks absence of parasitemia is recommended.[44] Azithromycin has been used as a replacement drug for clindamycin or quinine in cases of treatment failure or adverse reactions in severe infections with a successful outcome.[454647] Successful outcomes have also been reported in the treatment of cases with pentamidine–cotrimoxazole and atovaquone-proguanil combinations. Antimalarial drugs have been found to be ineffective against Babesia although certain in vitro studies report susceptibility to artemisinin derivatives.[484950]

The CDC has recommended preventive strategies, which basically target avoidance of tick bites/exposure particularly for immunosuppressed or splenectomized individuals since the infection is fulminant. The risk of exposure/bite is highest during the months of May-September and travel to endemic or tick-infested areas to be avoided.[1] Covering exposed areas with clothes, tucking of pants in shoes, skin application of various acaricides like N, N–diethylmetatoluamide, wearing light colored and permethrin-impregnated clothes, and careful examination for ticks in body after travel, particularly in children and pets were recommended to prevent tick exposure/bite.[51] Screening of blood donors using a combination of IFA and PCR has been effective in preventing TTB.[52] Currently, no vaccine is available for the prevention of babesiosis.

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Conflicts of interest

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