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.

Transfection of Babesia duncani: A Genetic Toolbox of this Pathogen to Advance Babesia Biology.

Human babesiosis is a tick-borne disease caused by Babesia pathogens. The disease, which presents with malaria-like symptoms, can be life-threatening, especially in individuals with weakened immune systems and the elderly. The worldwide prevalence of human babesiosis has been gradually rising, prompting alarm among public health experts. In other pathogens, genetic techniques have proven to be valuable tools for conducting functional studies to understand the importance of specific genes in development and pathogenesis as well as to validate novel cellular targets for drug discovery. Genetic manipulation methods have been established for several non-human Babesia and Theileria species and, more recently, have begun to be developed for human Babesia parasites. We have previously reported the development of a method for genetic manipulation of the human pathogen Babesia duncani. This method is based on positive selection using the hDHFR gene as a selectable marker, whose expression is regulated by the ef-1aB promoter, along with homology regions that facilitate integration into the gene of interest through homologous recombination. Herein, we provide a detailed description of the steps needed to implement this strategy in B. duncani to study gene function. It is anticipated that the implementation of this method will significantly improve our understanding of babesiosis and facilitate the development of novel and more effective therapeutic strategies for the treatment of human babesiosis. Key features This protocol provides an effective means of transfection of B. duncani, enabling genetic manipulation and editing to gain further insights into its biology and pathogenesis. The protocol outlined here for the electroporation of B. duncani represents an advancement over previous methods used for B. bovis [1]. Improvements include higher volume of culture used during the electroporation step and an enhancement in the number of electroporation pulses. These modifications likely enhance the efficiency of gene editing in B. duncani, allowing for quicker and more effective selection of transgenic parasites.

Babesia duncani Pyruvate Kinase Inhibitor Screening and Identification of Key Active Amino Acid Residues.

Babesia duncani (B. duncani), a protozoan parasite prevalent in North America, is a significant threat for human health. Given the regulatory role of pyruvate kinase I (PyK I) in glycolytic metabolism flux and ATP generation, PyK I has been considered the target for drug intervention for a long time. In this study, B. duncani PyK I (BdPyK I) was successfully cloned, expressed, and purified. Polyclonal antibodies were confirmed to recognize the native BdPyK I protein (56 kDa) using Western blotting. AlphaFold software predicted the three-dimensional structure of BdPyK I, and molecular docking with small molecules was conducted to identify potential binding sites of inhibitor on BdPyK I. Moreover, inhibitory effects of six inhibitors (tannic acid, apigenin, shikonin, PKM2 inhibitor, rosiglitazone, and pioglitazone) on BdPyK I were examined under the optimal enzymatic conditions of 3 mM PEP and 3 mM ADP, and significant activity reduction was found. Enzyme kinetics and growth inhibition assays further confirmed the reliability of these inhibitors, with PKM2 inhibitor, tannic acid, and apigenin exhibiting the highest selectivity index as specific inhibitors for B. duncani. Subsequently, key amino acid residues were mutated in both BdPyK I and Homo sapiens pyruvate kinase I (HPyK I), and two differential amino acid residues (isoleucine and phenylalanine) were identified between HPyK I and BdPyK I through PyK activity detection experiments. These findings lay foundation for understanding the role of PyK I in the growth and development of B. duncani, providing insights for babesiosis prevention and drug development.

A set of diagnostic tests for detection of active Babesia duncani infection.

OBJECTIVES: Human babesiosis is an emerging and potentially fatal tick-borne disease caused by intraerythrocytic parasites of the Babesia genus. Among these, Babesia duncani is particularly notable for causing severe and life-threatening illness in humans. Accurate diagnosis and effective disease management hinge on the detection of active B. duncani infections. While molecular assays are available to detect the parasite in blood, a reliable method for identifying biomarkers of active infection remains elusive. METHODS: We developed the first B. duncani antigen capture assays, targeting two immunodominant antigens, BdV234 and BdV38. These assays were validated using established in vitro and in vivo B. duncani infection models, and following drug treatment. RESULTS: The assays demonstrated no cross-reactivity with other species such as B. microti, B. divergens, Babesia MO1, or Plasmodium falciparum, and can detect as few as 115 infected erythrocytes/µl of blood. Screening of 1731 blood samples from various biorepositories, including samples previously identified as Lyme and/or B. microti-positive, as well as new specimens from wild mice, revealed no evidence of B. duncani infection or cross-reactivity. CONCLUSIONS: These assays hold significant promise for various applications, including point-of-care testing for the early detection of B. duncani in patients, field tests for screening reservoir hosts, and high-throughput screening of blood samples intended for transfusion.

Babeisa duncani infection alters gut microbiota profile in hamsters.

The genus Babesia includes parasites that can induce human and animal babesiosis, which are common in tropical and subtropical regions of the world. The gut microbiota has not been examined in hamsters infected by Babesia duncani. Red blood cells infected with B. duncani were injected into hamsters through intraperitoneal route. To evaluate the changes in gut microbiota, DNAs were extracted from small intestinal contents, acquired from hamsters during disease development. Then, the V4 region of the 16S rRNA gene of bacteria was sequenced using the Illumina sequencing platform. Gut microbiota alternation and composition were assessed according to the sequencing data, which were clustered with >97.0% sequence similarity to create amplicon sequence variants (ASVs). Bacteroidetes and Firmicutes were made up of the major components of the gut microbiota in all samples. The abundance of Bacteroidetes elevated after B. duncani infection than the B. duncani-free group, while Firmicutes and Desulfobacterota declined. Alpha diversity analysis demonstrated that the shown ASVs were substantially decreased in the highest parasitemia group than B. duncani-free and lower parasitemia groups. Potential biomarkers were discovered by Linear discriminant analysis Effect Size (LEfSe) analysis, which demonstrated that several bacterial families (including Muribaculaceae, Desulfovibrionaceae, Oscillospiraceae, Helicobacteraceae, Clostridia UGG014, Desulfovibrionaceae, and Lachnospiraceae) were potential biomarkers in B. duncani-infected hamsters. This research demonstrated that B. duncani infectious can modify the gut microbiota of hamsters.

Continuous In Vitro Culture of Babesia duncani in a Serum-Free Medium.

Human babesiosis is an emerging tick-borne disease, caused by haemoprotozoa genus of Babesia. Cases of transfusion-transmitted and naturally acquired Babesia infection have been reported worldwide in recent years and causing a serious public health problem. Babesia duncani is one of the important pathogens of human babesiosis, which seriously endangers human health. The in vitro culture systems of B. duncani have been previously established, and it requires fetal bovine serum (FBS) to support long-term proliferation. However, there are no studies on serum-free in vitro culture of B. duncani. In this study, we reported that B. duncani achieved long-term serum-free culture in VP-SFM AGTTM (VP-SFM) supplemented with AlbuMaxTM I. The effect of adding different dilutions of AlbuMaxTM I to VP-SFM showed that 2 mg/mL AlbuMaxTM I had the best B. duncani growth curve with a maximum percentage of parasitized erythrocytes (PPE) of over 40%, and it can be used for long-term in vitro culture of B. duncani. However, the commonly used 20% serum-supplemented medium only achieves 20% PPE. Clearly, VP-SFM with 2 mg/mL AlbuMaxTM I (VP-SFMA) is more suitable for the in vitro proliferation of B. duncani. VP-SFM supplemented with CD lipid mixture was also tested, and the results showed it could support the parasite growth at 1:100 dilution with the highest PPE of 40%, which is similar to that of 2 mg/mL AlbuMaxTM I. However, the CD lipid mixture was only able to support the in vitro culture of B. duncani for 8 generations, while VP-SFMA could be used for long-term culture. To test the pathogenicity, the VP-SFMA cultured B. duncani was also subjected to hamster infection. Results showed that the hamster developed dyspnea and chills on day 7 with 30% PPE before treatment, which is similar to the symptoms with un-cultured B. duncani. This study develops a unique and reliable basis for further understanding of the physiological mechanisms, growth characteristics, and pathogenesis of babesiosis, and provides good laboratory material for the development of drugs or vaccines for human babesiosis and possibly other parasitic diseases.

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)].

Establishment of a Transient and Stable Transfection System for Babesia duncani Using a Homologous Recombination Strategy.

Genetic modification provides an invaluable molecular tool to dissect the biology and pathogenesis of pathogens. However, no report is available about the genetic modification of Babesia duncani, a pathogen responsible for human babesiosis that is widespread in North America, suggesting the necessity to develop a genetic manipulation method to improve the strategies for studying and understanding the biology of protozoan pathogens. The establishment of a genetic modification method requires promoters, selectable markers, and reporter genes. Here, the double-copy gene elongation factor-1α (ef-1α) and its promoters were amplified by conventional PCR and confirmed by sequencing. We established a transient transfection system by using the ef-1αB promoter and the reporter gene mCherry and achieved stable transfection through homologous recombination to integrate the selection marker hDHFR-eGFP into the parasite genome. The potential of this genetic modification method was tested by knocking out the thioredoxin peroxidase-1 (TPX-1) gene, and under the drug pressure of 5 nM WR99210, 96.3% of the parasites were observed to express green fluorescence protein (eGFP) by flow cytometry at day 7 post-transfection. Additionally, the clone line of the TPX-1 knockout parasite was successfully obtained by the limiting dilution method. This study provided a transfection method for B. duncani, which may facilitate gene function research and vaccine development of B. duncani.

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.

Fluorescence In Situ Hybridization (FISH) Tests for Identifying Protozoan and Bacterial Pathogens in Infectious Diseases.

Diagnosing and treating many infectious diseases depends on correctly identifying the causative pathogen. Characterization of pathogen-specific nucleic acid sequences by PCR is the most sensitive and specific method available for this purpose, although it is restricted to laboratories that have the necessary infrastructure and finance. Microscopy, rapid immunochromatographic tests for antigens, and immunoassays for detecting pathogen-specific antibodies are alternative and useful diagnostic methods with different advantages and disadvantages. Detection of ribosomal RNA molecules in the cytoplasm of bacterial and protozoan pathogens by fluorescence in-situ hybridization (FISH) using sequence-specific fluorescently labelled DNA probes, is cheaper than PCR and requires minimal equipment and infrastructure. A LED light source attached to most laboratory light microscopes can be used in place of a fluorescence microscope with a UV lamp for FISH. A FISH test hybridization can be completed in 30 min at 37 °C and the whole test in less than two hours. FISH tests can therefore be rapidly performed in both well-equipped and poorly-resourced laboratories. Highly sensitive and specific FISH tests for identifying many bacterial and protozoan pathogens that cause disease in humans, livestock and pets are reviewed, with particular reference to parasites causing malaria and babesiosis, and mycobacteria responsible for tuberculosis.

Botanical Medicines Cryptolepis sanguinolenta, Artemisia annua, Scutellaria baicalensis, Polygonum cuspidatum, and Alchornea cordifolia Demonstrate Inhibitory Activity Against Babesia duncani.

Human babesiosis is a CDC reportable disease in the United States and is recognized as an emerging health risk in multiple parts of the world. The current treatment for human babesiosis is suboptimal due to treatment failures and unwanted side effects. Although Babesia duncani was first described almost 30 years ago, further research is needed to elucidate its pathogenesis and clarify optimal treatment regimens. Here, we screened a panel of herbal medicines and identified Cryptolepis sanguinolenta, Artemisia annua, Scutellaria baicalensis, Alchornea cordifolia, and Polygonum cuspidatum to have good in vitro inhibitory activity against B. duncani in the hamster erythrocyte model. Furthermore, we found their potential bioactive compounds, cryptolepine, artemisinin, artesunate, artemether, and baicalein, to have good activity against B. duncani, with IC50 values of 3.4 µM, 14 µM, 7.4 µM, 7.8 µM, and 12 µM, respectively, which are comparable or lower than that of the currently used drugs quinine (10 µM) and clindamycin (37 µM). B. duncani treated with cryptolepine and quinine at their respective 1×, 2×, 4× and 8× IC50 values, and by artemether at 8× IC50 for three days could not regrow in subculture. Additionally, Cryptolepis sanguinolenta 90% ethanol extract also exhibited no regrowth after 6 days of subculture at doses of 2×, 4×, and 8× IC50 values. Our results indicate that some botanical medicines and their active constituents have potent activity against B. duncani in vitro and may be further explored for more effective treatment of babesiosis.