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.

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.

Babesia duncani multi-omics identifies virulence factors and drug targets.

Babesiosis is a malaria-like disease in humans and animals that is caused by Babesia species, which are tick-transmitted apicomplexan pathogens. Babesia duncani causes severe to lethal infection in humans, but despite the risk that this parasite poses as an emerging pathogen, little is known about its biology, metabolic requirements or pathogenesis. Unlike other apicomplexan parasites that infect red blood cells, B. duncani can be continuously cultured in vitro in human erythrocytes and can infect mice resulting in fulminant babesiosis and death. We report comprehensive, detailed molecular, genomic, transcriptomic and epigenetic analyses to gain insights into the biology of B. duncani. We completed the assembly, 3D structure and annotation of its nuclear genome, and analysed its transcriptomic and epigenetics profiles during its asexual life cycle stages in human erythrocytes. We used RNA-seq data to produce an atlas of parasite metabolism during its intraerythrocytic life cycle. Characterization of the B. duncani genome, epigenome and transcriptome identified classes of candidate virulence factors, antigens for diagnosis of active infection and several attractive drug targets. Furthermore, metabolic reconstitutions from genome annotation and in vitro efficacy studies identified antifolates, pyrimethamine and WR-99210 as potent inhibitors of B. duncani to establish a pipeline of small molecules that could be developed as effective therapies for the treatment of human babesiosis.

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.

A Whole Genome Sequencing-Based Approach to Track down Genomic Variants in Itraconazole-Resistant Species of Aspergillus from Iran.

The antifungal resistance in non-fumigatus Aspergillus spp., as well as Aspergillus fumigatus, poses a major therapeutic challenge which affects the entire healthcare community. Mutation occurrence of cyp51 gene paralogs is the major cause of azole resistance in Aspergillus spp. To obtain a full map of genomic changes, an accurate scan of the entire length of the Aspergillus genome is necessary. In this study, using whole genome sequencing (WGS) technique, we evaluated the mutation in cyp51A, cyp51B, Cdr1B, AtrR, Hmg1, HapE and FfmA genes in different clinical isolates of Aspergillus fumigatus, Aspergillus niger, Aspergillus tubingensis, Aspergillus welwitschiae and Aspergillus terreus which responded to minimum inhibitory concentrations of itraconazole above 16 µg mL-1. We found different nonsynonymous mutations in the cyp51A, cyp51B, Cdr1B, AtrR, Hmg1, HapE and FfmA gene loci. According to our findings, Aspergillus species isolated from different parts of the world may represent different pattern of resistance mechanisms which may be revealed by WGS.

High-resolution crystal structure and chemical screening reveal pantothenate kinase as a new target for antifungal development.

Fungal infections are the leading cause of mortality by eukaryotic pathogens, with an estimated 150 million severe life-threatening cases and 1.7 million deaths reported annually. The rapid emergence of multidrug-resistant fungal isolates highlights the urgent need for new drugs with new mechanisms of action. In fungi, pantothenate phosphorylation, catalyzed by PanK enzyme, is the first step in the utilization of pantothenic acid and coenzyme A biosynthesis. In all fungi sequenced so far, this enzyme is encoded by a single PanK gene. Here, we report the crystal structure of a fungal PanK alone as well as with high-affinity inhibitors from a single chemotype identified through a high-throughput chemical screen. Structural, biochemical, and functional analyses revealed mechanisms governing substrate and ligand binding, dimerization, and catalysis and helped identify new compounds that inhibit the growth of several Candida species. The data validate PanK as a promising target for antifungal drug development.

BSA-PEI Nanoparticle Mediated Efficient Delivery of CRISPR/Cas9 into MDA-MB-231 Cells.

The discovery of bacterial-derived Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has revolutionized genome engineering and gene therapy due to its wide range of applications. One of the major challenging issues in CRISPR/Cas system is the lack of an efficient, safe, and clinically suitable delivery of the system's components into target cells. Here, we describe the development of polyethylenimine coated-bovine serum albumin nanoparticles (BSA-PEI NPs) for efficient delivery of CRISPR/Cas9 system in both DNA (px458 plasmid) and ribonucleoprotein (RNP) forms into MDA-MB-231 human breast cancer cell line. Our data showed that synthesized BSA-PEI (BP) NPs delivered plasmid px458 at concentrations of 0.15, 0.25, and 0.35 µg/µl with efficiencies of approximately 29.7, 54.8, and 84.1% into MDA-MB-231 cells, respectively. Our study demonstrated that Cas9/sgRNA RNP complex efficiently (~ 92.6%) delivered by BSA-PEI NPs into the same cells. Analysis of toxicity and biocompatibility of synthesized NPs on human red blood cells, MDA-MB-231 cells, and mice showed that the selected concentration (28 µg/µl) of BSA-PEI NPs for transfection had no remarkable toxicity effects. Thus, obtained results suggest BSA-PEI NPs as one of the most promising carrier for delivering CRISPR/Cas9 to target cells.

Plasmid-based CRISPR-Cas9 system efficacy for introducing targeted mutations in CD81 gene of MDA-MB-231 cell line.

INTRODUCTION: Breast cancer has been represented a challenging issue worldwide as it is one of the major leading causes of death among women. CD81 gene, a member of the tetraspanin protein family, has been associated with the development of human cancers. Genome editing technologies, particularly the CRISPR-Cas9 system, have shown rapid progress in gene function studies. In this study, we aimed to evaluate the ability of the CRISPR-Cas9 plasmid-based system to modify specific regions of the CD81 gene in the MDA-MB-231 breast cancer cell line. MATERIALS AND METHODS: Using bioinformatics database search, four different single guide RNAs (sgRNAs) to target exon 3 and exon 5 of the CD81 gene were designed. The intended sgRNAs sequences were cloned into the expression plasmid pSpCas9(BB)-2A-GFP (PX458) bearing sgRNA scaffold backbone, Cas9, and EGFP coding sequences, which was confirmed by colony PCR and sequencing. Transfection efficiency was determined by fluorescence microscopy and flow cytometry analysis. Gene editing efficiency was measured qualitatively and quantitatively using the T7E1 and TIDE software, respectively. RESULTS: Our data show that expression constructs were successfully introduced into MDA-MB-231 cells with an acceptable transfection efficiency. Two sgRNAs that were afforded to introduce significant mutations in their target regions were detected by TIDE software (p-value < 0.05). To the best of our knowledge, CD81 gene editing in these cells has been investigated for the first time in this study using the CRISPR/Cas9 technique. CONCLUSIONS: Taken together, our data show that the CRISPR-Cas9 system can change the genomic sequence in the target area of MDA-MB-231 cells. Along with previous studies, we propose forethought when using T7E1-based quantitative indel estimates, as comparing activities of multiple gRNAs with the T7E1 assay may lead to inaccurate conclusions. Instead, estimating non-homologous end-joining events (NHEJ) by Sanger sequencing and subsequent TIDE analysis is recommended.

Babesia microti Immunoreactive Rhoptry-Associated Protein-1 Paralogs Are Ancestral Members of the Piroplasmid-Confined RAP-1 Family.

Babesia, Cytauxzoon and Theileria are tick-borne apicomplexan parasites of the order Piroplasmida, responsible for diseases in humans and animals. Members of the piroplasmid rhoptry-associated protein-1 (pRAP-1) family have a signature cysteine-rich domain and are important for parasite development. We propose that the closely linked B. microti genes annotated as BMR1_03g00947 and BMR1_03g00960 encode two paralogue pRAP-1-like proteins named BmIPA48 and Bm960. The two genes are tandemly arranged head to tail, highly expressed in blood stage parasites, syntenic to rap-1 genes of other piroplasmids, and share large portions of an almost identical ~225 bp sequence located in their 5' putative regulatory regions. BmIPA48 and Bm960 proteins contain a N-terminal signal peptide, share very low sequence identity (<13%) with pRAP-1 from other species, and harbor one or more transmembrane domains. Diversification of the piroplasmid-confined prap-1 family is characterized by amplification of genes, protein domains, and a high sequence polymorphism. This suggests a functional involvement of pRAP-1 at the parasite-host interface, possibly in parasite adhesion, attachment, and/or evasion of the host immune defenses. Both BmIPA48 and Bm960 are recognized by antibodies in sera from humans infected with B. microti and might be promising candidates for developing novel serodiagnosis and vaccines.

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.

Recent Advances in Genome Editing Tools in Medical Mycology Research.

Manipulating fungal genomes is an important tool to understand the function of target genes, pathobiology of fungal infections, virulence potential, and pathogenicity of medically important fungi, and to develop novel diagnostics and therapeutic targets. Here, we provide an overview of recent advances in genetic manipulation techniques used in the field of medical mycology. Fungi use several strategies to cope with stress and adapt themselves against environmental effectors. For instance, mutations in the 14 alpha-demethylase gene may result in azole resistance in Aspergillusfumigatus strains and shield them against fungicide's effects. Over the past few decades, several genome editing methods have been introduced for genetic manipulations in pathogenic fungi. Application of restriction enzymes to target and cut a double-stranded DNA in a pre-defined sequence was the first technique used for cloning in Aspergillus and Candida. Genome editing technologies, including zinc-finger nucleases (ZFNs) and transcriptional activator-like effector nucleases (TALENs), have been also used to engineer a double-stranded DNA molecule. As a result, TALENs were considered more practical to identify single nucleotide polymorphisms. Recently, Class 2 type II Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9 technology has emerged as a more useful tool for genome manipulation in fungal research.

A knockdown of the herpes simplex virus type-1 gene in all-in-one CRISPR vectors.

INTRODUCTION: Herpes simplex virus type 1 (HSV-1) is a virus that causes serious human disease and establishes a long-term latent infection. The latent form of this virus has shown to be resistant to antiviral drugs. Clustered Regularly Interspace Short Palindromic Repeats (CRISPR), is an important tool in genome engineering and composed of guide RNA (gRNA) and Cas9 nuclease that makes an RNA-protein complex to digest exclusive target sequences implementation of gRNA. Moreover, CRISPR-Cas9 system effectively suppresses HSV-1 infection by knockout of some viral genes. MATERIALS AND METHODS: To survey the efficacy of Cas9 system on HSV-1 genome destruction, we designed several guide RNAs (gRNAs) that all packaged in one vector. Additionally, we performed a one-step restriction using BamHI and Esp3I enzymes. RESULTS: CRISPR/Cas9 system targeted against the gD gene of HSV-1 was transfected into HEK-AD cells that showed a significant reduction of HSV-1 infection by plaque assay and real-time PCR. CONCLUSION: The pCas-Guide-EF1a-GFP CRISPR vector can create a fast and efficient method for gRNA cloning by restriction enzymes (Esp3I (BsmBI) and BamHI). Therefore, the CRISPR/Cas9 system may be utilized for the screening of genes critical for the HSV-1 infection and developing new strategies for targeted therapy of viral infections caused by HSV-1.

An overview of applications of CRISPR-Cas technologies in biomedical engineering.

Clustered Regulatory Interspaced Short Palindromic Repeats (CRISPR) is one of the major genome editing systems and allows changing DNA levels of an organism. Among several CRISPR categories, the CRISPR-Cas9 system has shown a remarkable progression rate over its lifetime. Recently, other tools including CRISPR-Cas12 and CRISPR-Cas13 have been introduced. CRISPR-Cas9 system has played a key role in the industrial cell factory's production and improved our understanding of genome function. Additionally, this system has been used as one of the major genome editing systems for the diagnosis and treatment of several infectious and non-infectious diseases. In this review, we discuss CRISPR biology, its versatility, and its application in biomedical engineering.

The yeast pantothenate kinase Cab1 is a master regulator of sterol metabolism and of susceptibility to ergosterol biosynthesis inhibitors.

In fungi, ergosterol is an essential component of the plasma membrane. Its biosynthesis from acetyl-CoA is the primary target of the most commonly used antifungal drugs. Here, we show that the pantothenate kinase Cab1p, which catalyzes the first step in the metabolism of pantothenic acid for CoA biosynthesis in budding yeast (Saccharomyces cerevisiae), significantly regulates the levels of sterol intermediates and the activities of ergosterol biosynthesis-targeting antifungals. Using genetic and pharmacological analyses, we show that altered pantothenate utilization dramatically alters the susceptibility of yeast cells to ergosterol biosynthesis inhibitors. Genome-wide transcription and MS-based analyses revealed that this regulation is mediated by changes both in the expression of ergosterol biosynthesis genes and in the levels of sterol intermediates. Consistent with these findings, drug interaction experiments indicated that inhibition of pantothenic acid utilization synergizes with the activity of the ergosterol molecule-targeting antifungal amphotericin B and antagonizes that of the ergosterol pathway-targeting antifungal drug terbinafine. Our finding that CoA metabolism controls ergosterol biosynthesis and susceptibility to antifungals could set the stage for the development of new strategies to manage fungal infections and to modulate the potency of current drugs against drug-sensitive and -resistant fungal pathogens.

Evidence for vesicle-mediated antigen export by the human pathogen Babesia microti.

The apicomplexan parasite Babesia microti is the primary agent of human babesiosis, a malaria-like illness and potentially fatal tick-borne disease. Unlike its close relatives, the agents of human malaria, B. microti develops within human and mouse red blood cells in the absence of a parasitophorous vacuole, and its secreted antigens lack trafficking motifs found in malarial secreted antigens. Here, we show that after invasion of erythrocytes, B. microti undergoes a major morphogenic change during which it produces an interlacement of vesicles (IOV); the IOV system extends from the plasma membrane of the parasite into the cytoplasm of the host erythrocyte. We developed antibodies against two immunodominant antigens of the parasite and used them in cell fractionation studies and fluorescence and immunoelectron microscopy analyses to monitor the mode of secretion of B. microti antigens. These analyses demonstrate that the IOV system serves as a major export mechanism for important antigens of B. microti and represents a novel mechanism for delivery of parasite effectors into the host by this apicomplexan parasite.

High-throughput screening for phosphatidylserine decarboxylase inhibitors using a distyrylbenzene-bis-aldehyde (DSB-3)-based fluorescence assay.

Phosphatidylserine decarboxylases (PSDs) catalyze the decarboxylation of phosphatidylserine to generate phosphatidylethanolamine, a critical step in phospholipid metabolism in both prokaryotes and eukaryotes. Most PSDs are membrane-bound, and classical radioisotope-based assays for determining their activity in vitro are not suitable for high-throughput drug screening. The finding that the PkPSD from Plasmodium knowlesi can be purified in a soluble and active form and the recent development of a fluorescence-based distyrylbenzene-bis-aldehyde (DSB-3) assay to measure PSD activity in vitro have laid the groundwork for screening chemical libraries for PSD inhibitors. Using this assay, here we conducted a high-throughput screen of a structurally diverse 130,858-compound library against PkPSD. Further characterization of the hits identified in this screening yielded five PkPSD inhibitors with IC50 values ranging from 3.1 to 42.3 µm Lead compounds were evaluated against the pathogenic yeast Candida albicans in the absence or presence of exogenous ethanolamine, and YU253467 and YU254403 were identified as inhibiting both native C. albicans PSD mitochondrial activity and C. albicans growth, with an MIC50 of 22.5 and 15 µg/ml without ethanolamine and an MIC50 of 75 and 60 µg/ml with ethanolamine, respectively. Together, these results provide the first proof of principle for the application of DSB-3-based fluorescent readouts in high-throughput screening for PSD inhibitors. The data set the stage for future analyses to identify more selective and potent PSD inhibitors with antimicrobial or antitumor activities.

The nuclear hormone receptor NHR-86 controls anti-pathogen responses in C. elegans.

Nuclear hormone receptors (NHRs) are ligand-gated transcription factors that control adaptive host responses following recognition of specific endogenous or exogenous ligands. Although NHRs have expanded dramatically in C. elegans compared to other metazoans, the biological function of only a few of these genes has been characterized in detail. Here, we demonstrate that an NHR can activate an anti-pathogen transcriptional program. Using genetic epistasis experiments, transcriptome profiling analyses and chromatin immunoprecipitation-sequencing, we show that, in the presence of an immunostimulatory small molecule, NHR-86 binds to the promoters of immune effectors to activate their transcription. NHR-86 is not required for resistance to the bacterial pathogen Pseudomonas aeruginosa at baseline, but activation of NHR-86 by this compound drives a transcriptional program that provides protection against this pathogen. Interestingly, NHR-86 targets immune effectors whose basal regulation requires the canonical p38 MAPK PMK-1 immune pathway. However, NHR-86 functions independently of PMK-1 and modulates the transcription of these infection response genes directly. These findings characterize a new transcriptional regulator in C. elegans that can induce a protective host response towards a bacterial pathogen.

Insights into the evolution and drug susceptibility of Babesia duncani from the sequence of its mitochondrial and apicoplast genomes.

Babesia microti and Babesia duncani are the main causative agents of human babesiosis in the United States. While significant knowledge about B. microti has been gained over the past few years, nothing is known about B. duncani biology, pathogenesis, mode of transmission or sensitivity to currently recommended therapies. Studies in immunocompetent wild type mice and hamsters have shown that unlike B. microti, infection with B. duncani results in severe pathology and ultimately death. The parasite factors involved in B. duncani virulence remain unknown. Here we report the first known completed sequence and annotation of the apicoplast and mitochondrial genomes of B. duncani. We found that the apicoplast genome of this parasite consists of a 34 kb monocistronic circular molecule encoding functions that are important for apicoplast gene transcription as well as translation and maturation of the organelle's proteins. The mitochondrial genome of B. duncani consists of a 5.9 kb monocistronic linear molecule with two inverted repeats of 48 bp at both ends. Using the conserved cytochrome b (Cytb) and cytochrome c oxidase subunit I (coxI) proteins encoded by the mitochondrial genome, phylogenetic analysis revealed that B. duncani defines a new lineage among apicomplexan parasites distinct from B. microti, Babesia bovis, Theileria spp. and Plasmodium spp. Annotation of the apicoplast and mitochondrial genomes of B. duncani identified targets for development of effective therapies. Our studies set the stage for evaluation of the efficacy of these drugs alone or in combination against B. duncani in culture as well as in animal models.

Evidence for transmission of the zoonotic apicomplexan parasite Babesia duncani by the tick Dermacentor albipictus.

Babesiosis is a potentially fatal tick-borne zoonotic disease caused by a species complex of blood parasites that can infect a variety of vertebrates, particularly dogs, cattle, and humans. In the United States, human babesiosis is caused by two distinct parasites, Babesia microti and Babesia duncani. The enzootic cycle of B. microti, endemic in the northeastern and upper midwestern regions, has been well characterised. In the western United States, however, the natural reservoir host and tick vector have not been identified for B. duncani, greatly impeding efforts to understand and manage this zoonotic disease. Two and a half decades after B. duncani was first described in a human patient in Washington State, USA, we provide evidence that the enzootic tick vector is the winter tick, Dermacentor albipictus, and the reservoir host is likely the mule deer, Odocoileus hemionus. The broad, overlapping ranges of these two species covers a large portion of far-western North America, and is consistent with confirmed cases of B. duncani in the far-western United States.

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.