Tafenoquine-Atovaquone Combination Achieves Radical Cure and Confers Sterile Immunity in Experimental Models of Human Babesiosis.

Human babesiosis is a potentially fatal tick-borne disease caused by intraerythrocytic Babesia parasites. The emergence of resistance to recommended therapies highlights the need for new and more effective treatments. Here we demonstrate that the 8-aminoquinoline antimalarial drug tafenoquine inhibits the growth of different Babesia species in vitro, is highly effective against Babesia microti and Babesia duncani in mice and protects animals from lethal infection caused by atovaquone-sensitive and -resistant B. duncani strains. We further show that a combination of tafenoquine and atovaquone achieves cure with no recrudescence in both models of human babesiosis. Interestingly, elimination of B. duncani infection in animals following drug treatment also confers immunity to subsequent challenge. Altogether, the data demonstrate superior efficacy of tafenoquine plus atovaquone combination over current therapies for the treatment of human babesiosis and highlight its potential in providing protective immunity against Babesia following parasite clearance.

Babesia BdFE1 esterase is required for the anti-parasitic activity of the ACE inhibitor fosinopril.

Effective and safe therapies for the treatment of diseases caused by intraerythrocytic parasites are impeded by the rapid emergence of drug resistance and the lack of novel drug targets. One such disease is human babesiosis, which is a rapidly emerging tick-borne illness caused by Babesia parasites. In this study, we identified fosinopril, a phosphonate-containing, FDA-approved angiotensin converting enzyme (ACE) inhibitor commonly used as a prodrug for hypertension and heart failure, as a potent inhibitor of Babesia duncani parasite development within human erythrocytes. Cell biological and mass spectrometry analyses revealed that the conversion of fosinopril to its active diacid molecule, fosinoprilat, is essential for its antiparasitic activity. We show that this conversion is mediated by a parasite-encoded esterase, BdFE1, which is highly conserved among apicomplexan parasites. Parasites carrying the L238H mutation in the active site of BdFE1 failed to convert the prodrug to its active moiety and became resistant to the drug. Our data set the stage for the development of this class of drugs for the therapy of vector-borne parasitic diseases.

A novel and low-cost cross-priming amplification assay for rapid detection of Babesia duncani infection.

Babesia duncani, responsible for human babesiosis, is one of the most important tick-borne intraerythrocytic pathogens. Traditionally, babesiosis is definitively diagnosed by detecting parasite DNA in blood samples and examining Babesia parasites in Giemsa-stained peripheral blood smears. Although these techniques are valuable for determining Babesia duncani, they are often time-consuming and laborious. Therefore, developing rapid and reliable B. duncani identification assays is essential for subsequent epidemiological investigations and prevention and control. In this study, a cross-priming amplification (CPA) assay was developed, combined with a vertical flow visualization strip, to rapidly and accurately detect B. duncani infection. The detection limit of this method was as low as 0.98 pg/µl of genomic DNA from B. duncani merozoites per reaction at 59 °C for 60 min. There were no cross-reactions between B. duncani and other piroplasms infective to humans and mammals. A total of 592 blood samples from patients bitten by ticks and experimental infected hamsters were accurately assessed using CPA assay. The average cost of the CPA assay is as low as approximately $ 0.2 per person. These findings indicate that the CPA assay may therefore be a rapid screening tool for detection B. duncani infection, based on its accuracy, speed, and cost-effectiveness, particularly in resource-limited regions with a high prevalence of human babesiosis.

Comparative genomic analysis of Babesia duncani responsible for human babesiosis.

BACKGROUND: Human babesiosis, caused by parasites of the genus Babesia, is an emerging and re-emerging tick-borne disease that is mainly transmitted by tick bites and infected blood transfusion. Babesia duncani has caused majority of human babesiosis in Canada; however, limited data are available to correlate its genomic information and biological features. RESULTS: We generated a B. duncani reference genome using Oxford Nanopore Technology (ONT) and Illumina sequencing technology and uncovered its biological features and phylogenetic relationship with other Apicomplexa parasites. Phylogenetic analyses revealed that B. duncani form a clade distinct from B. microti, Babesia spp. infective to bovine and ovine species, and Theileria spp. infective to bovines. We identified the largest species-specific gene family that could be applied as diagnostic markers for this pathogen. In addition, two gene families show signals of significant expansion and several genes that present signatures of positive selection in B. duncani, suggesting their possible roles in the capability of this parasite to infect humans or tick vectors. CONCLUSIONS: Using ONT sequencing and Illumina sequencing technologies, we provide the first B. duncani reference genome and confirm that B. duncani forms a phylogenetically distinct clade from other Piroplasm parasites. Comparative genomic analyses show that two gene families are significantly expanded in B. duncani and may play important roles in host cell invasion and virulence of B. duncani. Our study provides basic information for further exploring B. duncani features, such as host-parasite and tick-parasite interactions.

Babesia duncani as a Model Organism to Study the Development, Virulence, and Drug Susceptibility of Intraerythrocytic Parasites In Vitro and In Vivo.

Human babesiosis is a malaria-like illness caused by tick-borne intraerythrocytic Babesia parasites of the Apicomplexa phylum. Whereas several species of Babesia can cause severe disease in humans, the ability to propagate Babesia duncani both in vitro in human erythrocytes and in mice makes it a unique pathogen to study Babesia biology and pathogenesis. Here we report an optimized B. duncani in culture-in mouse (ICIM) model that combines continuous in vitro culture of the parasite with a precise model of lethal infection in mice. We demonstrate that B. duncani-infected erythrocytes as well as free merozoites can cause lethal infection in C3H/HeJ mice. Highly reproducible parasitemia and survival outcomes could be established using specific parasite loads in different mouse genetic backgrounds. Using the ICIM model, we discovered 2 new endochin-like quinolone prodrugs (ELQ-331 and ELQ-468) that alone or in combination with atovaquone are highly efficacious against B. duncani and Babesia microti.

Nested qPCR assay to detect Babesia duncani infection in hamsters and humans.

Human babesiosis is caused by Babesia duncani that is transmitted through tick bites, blood transfusions, and transplacental transmission. Despite its health burden, diagnostic assays for this pathogen are either unsuitable for clinical applications or have a low detection efficiency; therefore, it remains undetected during transfusion and utilization of blood and blood-component transfusions. This study used a molecular approach via nested quantitative polymerase chain reaction (qPCR) by designing primers and probes corresponding to the variable regions of B. duncani 18S rRNA gene to specifically detect B. duncani DNA in experimentally infected LVG Golden Syrian hamster (n = 70) and human (n = 492; tick bite patients from Gansu Province, China) blood samples. Moreover, comparative analyses of this technique with previously reported nested PCR and microscopy were conducted. The newly optimized diagnostic technique exhibited no cross-reactivity with genomic DNA or plasmids containing the 18S rRNA gene of other zoonotically important Babesia spp., including B. microti, B. divergens, B. crassa, and B. motasi Hebei. The detection limit of nested qPCR was approximately one plasmid copy in 20 µL or one infected red blood cell in 200 µL whole blood. The specificity and sensitivity of the method were 100% and 98.6%, respectively. Comparative analyses revealed that nested qPCR detected B. duncani had relatively higher efficacy and specificity than microscopic examination and nested PCR. The 492 human blood samples were negative for B. duncani infection. Thus, the present study provides an improved diagnostic assay for the efficient and effective detection and analysis of B. duncani infections and its prevalence in infection-prone areas.

Effective Therapy Targeting Cytochrome bc1 Prevents Babesia Erythrocytic Development and Protects from Lethal Infection.

An effective strategy to control blood-borne diseases and prevent outbreak recrudescence involves targeting conserved metabolic processes that are essential for pathogen viability. One such target for Plasmodium and Babesia, the infectious agents of malaria and babesiosis, respectively, is the mitochondrial cytochrome bc1 protein complex, which can be inhibited by endochin-like quinolones (ELQ) and atovaquone. We used the tick-transmitted and culturable blood-borne pathogen Babesia duncani to evaluate the structure-activity relationship, safety, efficacy, and mode of action of ELQs. We identified a potent and highly selective ELQ prodrug (ELQ-502), which, alone or in combination with atovaquone, eliminates B. microti and B. duncani infections in vitro and in mouse models of parasitemia and lethal infection. The strong efficacy at low dose, excellent safety, bioavailability, and long half-life of this experimental therapy make it an ideal clinical candidate for the treatment of human infections caused by Babesia and its closely related apicomplexan parasites.

Development of a Multiplex Bead Assay To Detect Immunoglobulin G Antibodies to Babesia duncani in Human Serum.

Babesia duncani is the causative agent of babesiosis in the western United States. The indirect fluorescent antibody (IFA) assay is the diagnostic test of choice for detection of B. duncani-specific antibodies. However, this test requires parasitized red blood cells harvested from infected hamsters, and test results are often difficult to interpret. To simplify serological testing for B. duncani, a proteomics approach was employed to identify candidate immunodiagnostic antigens. Several proteins were identified by electrospray ionization mass spectrometric analysis, and four recombinant protein constructs were expressed and used in a multiplex bead assay (MBA) to detect B. duncani-specific antibodies. Two antigens, AAY83295.1 and AAY83296.1, performed well with high sensitivities and specificities. AAY83295.1 had a higher sensitivity (100%) but lower specificity (89%) than AAY83296.1, which had a sensitivity of 90% and a specificity of 96%. Combining these two antigens did not improve the performance of the assay. This MBA could be useful for diagnosis, serosurveillance, and blood donor screening for B. duncani infection.

Establishment of a continuous in vitro culture of Babesia duncani in human erythrocytes reveals unusually high tolerance to recommended therapies.

Human babesiosis is an emerging tick-borne disease caused by apicomplexan parasites of the genus Babesia Clinical cases caused by Babesia duncani have been associated with high parasite burden, severe pathology, and death. In both mice and hamsters, the parasite causes uncontrolled fulminant infections, which ultimately lead to death. Resolving these infections requires knowledge of B. duncani biology, virulence, and susceptibility to anti-infectives, but little is known and further research is hindered by a lack of relevant model systems. Here, we report the first continuous in vitro culture of B. duncani in human red blood cells. We show that during its asexual cycle within human erythrocytes, B. duncani develops and divides to form four daughter parasites with parasitemia doubling every ∼22 h. Using this in vitro culture assay, we found that B. duncani has low susceptibility to the four drugs recommended for treatment of human babesiosis, atovaquone, azithromycin, clindamycin, and quinine, with IC50 values ranging between 500 nm and 20 µm These data suggest that current practices are of limited effect in treating the disease. We anticipate this new disease model will set the stage for a better understanding of the biology of this parasite and will help guide better therapeutic strategies to treat B. duncani-associated babesiosis.

In vitro cultivation of Babesia duncani (Apicomplexa: Babesiidae), a zoonotic hemoprotozoan, using infected blood from Syrian hamsters (Mesocricetus auratus).

Human babesiosis, a tick-borne disease similar to malaria, is most often caused by the hemoprotozoans Babesia divergens in Europe, and Babesia microti and Babesia duncani in North America. Babesia microti is the best documented and causes more cases of human babesiosis annually than all other agents combined. Although the agents that cause human babesiosis are considered high-risk pathogens in transfusion medicine, federally licensed diagnostics are lacking for B. duncani in both the USA and Canada. Thus, there has been a need to develop and validate diagnostics specifically for this pathogen. In this study, B. duncani (WA1 isolate) was cultivated in vitro from Syrian hamster (Mesocricetus auratus) infected blood. We hypothesized HL-1 media with supplements would result in B. duncani propagating at higher levels in culture than supplemented M199 similar to the medium the parasite was originally cultivated with in 1994. We were unable to recreate Thomford's cultivation results with the M199 medium but supplemented HL-1 medium was able to successfully establish continuous culture. We further hypothesized that RBC from species other than hamsters would support B. duncani in vitro. However, rat, mouse, horse, and cow RBC did not support continuous culture of the parasite. Culture stocks of B. duncani were deposited at BEI Resources and are now commercially available to the scientific community to further research. The cultured parasite developed in this study was instrumental in the adaptation of B. duncani continuous culture to human RBC.