A Set of Diagnostic Tests for Detection of Active Babesia duncani Infection.

Human babesiosis is a rapidly emerging and potentially fatal tick-borne disease caused by intraerythrocytic apicomplexan parasites of the Babesia genus. Among the various species of Babesia that infect humans, B. duncani has been found to cause severe and life-threatening infections. Detection of active B. duncani infection is critical for accurate diagnosis and effective management of the disease. While molecular assays for the detection of B. duncani infection in blood are available, a reliable strategy to detect biomarkers of active infection has not yet been developed. Here, we report the development of the first B. duncani antigen capture assays that rely on the detection of two B. duncani -exported immunodominant antigens, BdV234 and BdV38. The assays were validated using blood samples from cultured parasites in human erythrocytes and B. duncani -infected laboratory mice at different parasitemia levels and following therapy. The assays display high specificity with no cross-reactivity with B. microti , B. divergens , Babesia MO1, or P. falciparum. The assay also demonstrates high sensitivity, detecting as low as 115 infected erythrocytes/µl of blood. Screening of 1,731 blood samples from diverse biorepositories, including previously identified Lyme and/or B. microti positive human samples and new specimens from field mice, showed no evidence of B. duncani infection in these samples. The assays could be useful in diverse diagnostic scenarios, including point-of-care testing for early B. duncani infection detection in patients, field tests for screening reservoir hosts, and high-throughput screening such as blood collected for transfusion. Short summary: We developed two ELISA-based assays, BdACA38 and BdACA234, for detecting B. duncani , a potentially fatal tick-borne parasite causing human babesiosis. The assays target two immunodominant antigens, BdV234 and BdV38, demonstrating high specificity (no cross-reactivity with other Babesia species or Plasmodium falciparum ) and sensitivity (detecting as low as 115 infected erythrocytes/µl). The assays were validated using in vitro-cultured parasites and infected mice. Screening diverse blood samples showed no evidence of B. duncani active infection among 1,731 human and field mice blood samples collected from the north-eastern, midwestern, and western US. These assays offer potential in diverse diagnostic scenarios, including early patient detection, reservoir animal screening, and transfusion-transmitted babesiosis prevention.

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.

Epitope profiling of monoclonal antibodies to the immunodominant antigen BmGPI12 of the human pathogen Babesia microti.

The significant rise in the number of tick-borne diseases represents a major threat to public health worldwide. One such emerging disease is human babesiosis, which is caused by several protozoan parasites of the Babesia genus of which B. microti is responsible for most clinical cases reported to date. Recent studies have shown that during its intraerythrocytic life cycle, B. microti exports several antigens into the mammalian host using a novel vesicular-mediated secretion mechanism. One of these secreted proteins is the immunodominant antigen BmGPI12, which has been demonstrated to be a reliable biomarker of active B. microti infection. The major immunogenic determinants of this antigen remain unknown. Here we provide a comprehensive molecular and serological characterization of a set of eighteen monoclonal antibodies developed against BmGPI12 and a detailed profile of their binding specificity and suitability in the detection of active B. microti infection. Serological profiling and competition assays using synthetic peptides identified five unique epitopes on the surface of BmGPI12 which are recognized by a set of eight monoclonal antibodies. ELISA-based antigen detection assays identified five antibody combinations that specifically detect the secreted form of BmGPI12 in plasma samples from B. microti-infected mice and humans but not from other Babesia species or P. falciparum.

Specific and Sensitive Diagnosis of Babesia microti Active Infection Using Monoclonal Antibodies to the Immunodominant Antigen BmGPI12.

The apicomplexan pathogen Babesia microti is responsible for most cases of human babesiosis worldwide. The disease, which presents as a malaria-like illness, is potentially fatal in immunocompromised or elderly patients, making the need for its accurate and early diagnosis an urgent public health concern. B. microti is transmitted primarily by Ixodes ticks but can also be transmitted via blood transfusion. The parasite completes its asexual reproduction in the host red blood cell, where each invading merozoite develops and multiplies to produce four daughter parasites. While various techniques, such as microscopy, PCR, and indirect fluorescence, have been used over the years for babesiosis diagnosis, detection of the secreted B. microti immunodominant antigen BmGPI12 using specific polyclonal antibodies was found to be the most effective method for the diagnosis of active infection and for evaluation of clearance following drug treatment. Here, we report the development of a panel of 16 monoclonal antibodies against BmGPI12. These antibodies detected secreted BmGPI12 in the plasma of infected humans. Antigen capture assays identified a combination of two monoclonal antibodies, 4C8 and 1E11, as a basis for a monoclonal antibody-based BmGPI12 capture assay (mGPAC) to detect active B. microti infection. Using a collection of 105 previously characterized human plasma samples, the mGPAC assay showed 97.1% correlation with RNA-based PCR (transcription-mediated amplification [TMA]) for positive and negative samples. The mGPAC assay also detected BmGPI12 in the plasma of six babesiosis patients at the time of diagnosis but not in three matched posttreatment samples. The mGPAC assay could thus be used alone or in combination with other assays for accurate detection of active B. microti infection.

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.

An Alternative Culture Medium for Continuous In Vitro Propagation of the Human Pathogen Babesia duncani in Human Erythrocytes.

Continuous propagation of Babesia duncani in vitro in human erythrocytes and the availability of a mouse model of B. duncani lethal infection make this parasite an ideal model to study Babesia biology and pathogenesis. Two culture media, HL-1 and Claycomb, with proprietary formulations are the only culture media known to support the parasite growth in human erythrocytes; however, the HL-1 medium has been discontinued and the Claycomb medium is often unavailable leading to major interruptions in the study of this pathogen. To identify alternative media conditions, we evaluated the growth of B. duncani in various culture media with well-defined compositions. We report that the DMEM-F12 culture medium supports the continuous growth of the parasite in human erythrocytes to levels equal to those achieved in the HL-1 and Claycomb media. We generated new clones of B. duncani from the parental WA-1 clinical isolate after three consecutive subcloning events in this medium. All clones showed a multiplication rate in vitro similar to that of the WA-1 parental isolate and cause fatal infection in C3H/HeJ mice. The culture medium, which can be readily reconstituted from its individual components, and the tools and resources developed here will facilitate the study of B. duncani.

Babesia duncani in Culture and in Mouse (ICIM) Model for the Advancement of Babesia Biology, Pathogenesis, and Therapy.

Babesiosis is a tick-borne disease caused by pathogens belonging to the genus Babesia. In humans, the disease presents as a malaria-like illness and can be fatal in immunocompromised and elderly people. In the past few years, human babesiosis has been a rising concern worldwide. The disease is transmitted through tick bite, blood transfusion, and transplacentally in rare cases, with several species of Babesia causing human infection. Babesia microti, Babesia duncani, and Babesia divergens are of particular interest because of their important health impact and amenability to research inquiries. B. microti, the most commonly reported Babesia pathogen infecting humans, can be propagated in immunocompetent and immunocompromised mice but so far has not been successfully continuously propagated in vitro in human red blood cells (hRBCs). Conversely, B. divergens can be propagated in vitro in human red blood cells but lacks a mouse model to study its virulence. Recent studies have highlighted the uniqueness of B. duncani as an ideal model organism to study intraerythrocytic parasitism in vitro and in vivo. An optimized B. duncani in culture and in mouse (ICIM) model has recently been described, combining long-term continuous in vitro culture of the parasite in hRBCs with an animal model of parasitemia (P) and lethal infection in C3H/HeJ mice. Here, we provide a detailed protocol for the use of the B. duncani ICIM model in research. This model provides a unique and sound foundation to gain further insights into the biology, pathogenesis, and virulence of Babesia and other intraerythrocytic parasites, and has been validated as an efficient system to evaluate novel strategies for the treatment of human babesiosis and possibly other parasitic diseases. This protocol was validated in: J Infect Dis (2022), DOI: 10.1093/infdis/jiac181 Graphical abstract ICIM model [Adapted and modified from Pal et al. (2022)].

Evidence for a Conserved Function of Eukaryotic Pantothenate Kinases in the Regulation of Mitochondrial Homeostasis and Oxidative Stress.

Human PANK1, PANK2, and PANK3 genes encode several pantothenate kinase isoforms that catalyze the phosphorylation of vitamin B5 (pantothenic acid) to phosphopantothenate, a critical step in the biosynthesis of the major cellular cofactor, Coenzyme A (CoA). Mutations in the PANK2 gene, which encodes the mitochondrial pantothenate kinase (PanK) isoform, have been linked to pantothenate-kinase associated neurodegeneration (PKAN), a debilitating and often fatal progressive neurodegeneration of children and young adults. While the biochemical properties of these enzymes have been well-characterized in vitro, their expression in a model organism such as yeast in order to probe their function under cellular conditions have never been achieved. Here we used three yeast mutants carrying missense mutations in the yeast PanK gene, CAB1, which are associated with defective growth at high temperature and iron, mitochondrial dysfunction, increased iron content, and oxidative stress, to assess the cellular function of human PANK genes and functional conservation of the CoA-controlled processes between humans and yeast. Overexpression of human PANK1 and PANK3 in these mutants restored normal cellular activity whereas complementation with PANK2 was partial and could only be achieved with an isoform, PanK2mtmΔ, lacking the mitochondrial transit peptide. These data, which demonstrate functional conservation of PanK activity between humans and yeast, set the stage for the use of yeast as a model system to investigate the impact of PKAN-associated mutations on the metabolic pathways altered in this disease.

Cytochrome b Drug Resistance Mutation Decreases Babesia Fitness in the Tick Stages But Not the Mammalian Erythrocytic Cycle.

Human babesiosis is an emerging tick-borne malaria-like illness caused by Babesia parasites following their development in erythrocytes. Here, we show that a mutation in the Babesia microti mitochondrial cytochrome b (Cytb) that confers resistance to the antibabesial drug ELQ-502 decreases parasite fitness in the arthropod vector. Interestingly, whereas the mutant allele does not affect B. microti fitness during the mammalian blood phase of the parasite life cycle and is genetically stable as parasite burden increases, ELQ-502-resistant mutant parasites developing in the tick vector are genetically unstable with a high rate of the wild-type allele emerging during the nymphal stage. Furthermore, we show that B. microti parasites with this mutation are transmitted from the tick to the host, raising the possibility that the frequency of Cytb resistance mutations may be decreased by passage through the tick vector, but could persist in the environment if present when ticks feed.

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.