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.

Confirmed Case of Autochthonous Human Babesiosis, Hungary.

We report a case of autochthonous human babesiosis in Hungary, confirmed by PCR and partial sequencing of the Babesia spp. 18S rRNA gene. Babesiosis should be considered during the differential diagnosis of febrile illnesses, and peripheral blood smears to detect Babesia spp. should be part of the routine clinical workup.

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.

Extraordinary high level of propagation of Babesia divergens in severe human babesiosis.

Babesias are obligate apicomplexan parasites that affect the red blood cells (RBCs) of animals. Humans can serve as accidental hosts for them. Asexual reproduction of a parasite occurs in a vertebrate host through asynchronous binary fission, yielding a complex pleomorphic population of intraerythrocytic forms. In natural hosts (Bos taurus), paired pyriforms ('figure 8') of Babesia divergens are usual, but tetrads ('Maltese Cross') are very rare (only in 0.02% infected erythrocytes); in humans, however, up to 5% of infected erythrocytes show tetrads. The current study shows that B. divergens proliferating in an accidental human host can promote extraordinarily high level of fission. This phenomenon is expressed as the simultaneous division of the parasite into 6 and possibly a greater number of merozoites, forming a 'daisy head' (vs the usual 2, less often 4 merozoites). Reproduction is possible without egressing merozoites from the erythrocyte, which results in multi-occupancy of an RBC (≥5 parasites per RBC). An unusually high polyparasitism ­ up to 14 parasites developed in the affected erythrocytes ­ was observed. This phenomenon is rare in natural hosts (usually ≤5), but when B. divergens is cultured in vitro it can be 10­12.

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.

A Multiplex PCR assay for a differential diagnostic of rickettsiosis, Lyme disease and scrub typhus.

BACKGROUND & OBJECTIVES: Coexistence of tick-borne diseases in some regions in Latin America makes the diagnosis difficult due to shared initial signs and symptoms. Rickettsiosis, Lyme disease and recently, scrub typhus are gaining more importance. The objective of this study is to develop a multiplex-PCR assay for a differential diagnosis of rickettsiosis, Lyme disease and scrub typhus. METHODS: By using bibliographic and bioinformatic analysis, we identify candidate regions to perform the multiplex- PCR assay for Rickettsia sp., Borrelia burgdorferi and Orientia tsutsugamushi as well as identify optimal melting temperature and sensibility analysis. RESULTS: We identified specific primer pairs for Rickettsia sp, Borrelia burgdorferi and Orientia tsutsugamushi with different PCR fragment length but a common melting temperature, 58°C. INTERPRETATION & CONCLUSION: We successfully developed a Multiplex PCR assay for differential diagnosis of rickettsiosis, Lyme disease and scrub typhus that could be a rapid and easy option in clinical and epidemiological practice.

Transfusion-associated babesiosis in China: A case report.

Babesiosis, a novel zoonosis, is endemic in the Northeast and Midwest United States. This disease is primarily transmitted by ticks and less commonly transmitted through blood transfusion. Here, we present a case of human babesiosis of unknown etiology. The patient may have been infected through blood transfusion. This patient had fever for more than 1 month, accompanied by fatigue, anemia, jaundice, and other symptoms. Clinical improvement was unsatisfactory with antibiotics. Subsequently, peripheral blood smears showed many circulating forms of parasites,morphologically consistent with Babesia in red blood cells. Gene sequencing suggested Babesia microti. We treated the patient with azithromycin combined with other symptomatic supportive treatment. Finally, the patient recovered and was discharged. The intensity of babesiosis ranges from mild to severe and can be fatal, so early diagnosis and treatment are warranted.

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.

Human Babesiosis in China: a systematic review.

Human babesiosis, a worldwide emerging tick-borne disease, is caused by the intraerythrocytic apicomplexan parasite, babesia. In recent years, the number of infected patients globally has continued to rise, and thus human babesiosis poses a significant public health threat. Therefore, stronger initiatives should be undertaken to prevent further spread and development of this disease. In the present review, we summarize the epidemiology of reported human babesiosis cases in China from 1993 until now. The data show that Babesia microti is the dominant species causing human babesiosis in China and has led to more than 100 human infections thus far, where Babesia crassa-like is the second-most common. Moreover, Guangxi province is the second-most infected area after the Heilongjiang province. We also review the babesia life cycle, manifestation, diagnosis, and treatment. Additionally, we discuss babesiosis prevention strategies to raise public awareness, and also provide suggestions for improved babesiosis control.

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.

[Laboratory diagnostic of human babesiosis.]

Human babesiosis caused by parasitic protozoan Babesia spp. is sporadic zoonotic vector-borne infection. The course of babesiosis and prognosis depend on the type of pathogen and on the patient's immunological status. Significance this disease is a severe, often fatal course with immunocompromissed patients resembling complicated falciparum malaria. In Europe to date, more than 50 cases of confirmed human babesiosis have been reported in most cases caused by Babesia divergens. Possible there are unrecognized cases. Pathogen is an obligate intraerythrocyte parasite of vertebrate animals. The organism is transmitted from animal to man through bite of Ixodidae tick. Asexual reproduction of the parasite occurs in a vertebrate host. The pathogenesis of babesiosis is caused by the destruction of host cells. Intensive haemolysis of red blood cells leads to the development of haemolytic anemia, haematuria, jaundice, and polyorgan failure may develop. The clinical manifestations of the disease are nonspecific. Detection of intraerythrocyte parasites in blood smears stained Gimsa-Romanovsky confirms the proposed diagnosis. Blood smears and some laboratory signs from fatal cases were analyzed in the Reference-centre of E. I. Martsinovskii Institute. Original microphotographs B. divergens are shown. The main morphological forms of the parasite are shown. In addition to the well-known tetrades of parasites «Maltese Cross¼, for the first time, the parasites dividing into 6 interconnected trophozoites - "sextet" - were found. Originally, the invasion of Babesia in a normoblast is shown. An unusually high multiple invasion (14 parasites) of erythrocytes is noted. Because the patients, initially, were incorrectly diagnosed with malaria, the differential diagnosis of Babesia with Plasmodium is described step-by-step. It is important, since the treatment with antimalarial drugs is ineffective. Deviation laboratory signs are discussed. Complex morphological characteristics allowed us to speciated the parasites as B. divergens. DNA was detected in the sample with specific primers Bab di hsp70F/Bab di hsp70R and the probe Bab di hsp70P. The sequence demonstrated 99-100% and 98% similarity to the 18S rRNA gene fragment of B. divergence and Babesia venatorum, respectively. Molecular biological and serological methods of laboratory diagnosis of babesiosis are considered.

Misdiagnosis of Babesiosis as Malaria, Equatorial Guinea, 2014.

We report a case of babesiosis, caused by Babesia microti, in a missionary who worked in Equatorial Guinea but also visited rural Spain. The initial diagnosis, based on clinical features and microscopy, was malaria. The patient's recovery was delayed until she received appropriate treatment for babesiosis.

Transmission of Babesia microti Parasites by Solid Organ Transplantation.

Babesia microti, an intraerythrocytic parasite, is tickborne in nature. In contrast to transmission by blood transfusion, which has been well documented, transmission associated with solid organ transplantation has not been reported. We describe parasitologically confirmed cases of babesiosis diagnosed ≈8 weeks posttransplantation in 2 recipients of renal allografts from an organ donor who was multiply transfused on the day he died from traumatic injuries. The organ donor and recipients had no identified risk factors for tickborne infection. Antibodies against B. microti parasites were not detected by serologic testing of archived pretransplant specimens. However, 1 of the organ donor's blood donors was seropositive when tested postdonation and had risk factors for tick exposure. The organ donor probably served as a conduit of Babesia parasites from the seropositive blood donor to both kidney recipients. Babesiosis should be included in the differential diagnosis of unexplained fever and hemolytic anemia after blood transfusion or organ transplantation.

Monitoring human babesiosis emergence through vector surveillance New England, USA.

Human babesiosis is an emerging tick-borne disease caused by the intraerythrocytic protozoan Babesia microti. Its geographic distribution is more limited than that of Lyme disease, despite sharing the same tick vector and reservoir hosts. The geographic range of babesiosis is expanding, but knowledge of its range is incomplete and relies exclusively on reports of human cases. We evaluated the utility of tick-based surveillance for monitoring disease expansion by comparing the ratios of the 2 infections in humans and ticks in areas with varying B. microti endemicity. We found a close association between human disease and tick infection ratios in long-established babesiosis-endemic areas but a lower than expected incidence of human babesiosis on the basis of tick infection rates in new disease-endemic areas. This finding suggests that babesiosis at emerging sites is underreported. Vector-based surveillance can provide an early warning system for the emergence of human babesiosis.

Insights into the evolution, virulence and speciation of Babesia MO1 and Babesia divergens through multiomics analyses.

Babesiosis, caused by protozoan parasites of the genus Babesia, is an emerging tick-borne disease of significance for both human and animal health. Babesia parasites infect erythrocytes of vertebrate hosts where they develop and multiply rapidly to cause the pathological symptoms associated with the disease. The identification of new Babesia species underscores the ongoing risk of zoonotic pathogens capable of infecting humans, a concern amplified by anthropogenic activities and environmental changes. One such pathogen, Babesia MO1, previously implicated in severe cases of human babesiosis in the United States, was initially considered a subspecies of B. divergens, the predominant agent of human babesiosis in Europe. Here we report comparative multiomics analyses of B. divergens and B. MO1 that offer insight into their biology and evolution. Our analysis shows that despite their highly similar genomic sequences, substantial genetic and genomic divergence occurred throughout their evolution resulting in major differences in gene functions, expression and regulation, replication rates and susceptibility to antiparasitic drugs. Furthermore, both pathogens have evolved distinct classes of multigene families, crucial for their pathogenicity and adaptation to specific mammalian hosts. Leveraging genomic information for B. MO1, B. divergens, and other members of the Babesiidae family within Apicomplexa provides valuable insights into the evolution, diversity, and virulence of these parasites. This knowledge serves as a critical tool in preemptively addressing the emergence and rapid transmission of more virulent strains.

Transmission risk evaluation of transfusion blood containing low-density Babesia microti.

Background: Babesia is a unique apicomplexan parasite that specifically invades and proliferates in red blood cells and can be transmitted via blood transfusion, resulting in transfusion-transmitted babesiosis. However, detecting Babesia in blood before transfusion has not received enough attention, and the risk of transfusing blood containing a low density of Babesia microti (B. microti) is unclear, possibly threatening public health and wellness. Purpose: This study aimed to determine the lower detection limit of B. microti in blood and to evaluate the transmission risk of blood transfusion containing low-density B. microti. Methods: Infected BALB/c mouse models were established by transfusing infected whole blood with different infection rates and densities of B. microti. Microscopic examination, nested Polymerase Chain Reaction (nested PCR), and an enzyme-linked immunosorbent assay (ELISA) were used to evaluate the infection status of the mouse models. Meanwhile, the nested PCR detection limit of B. microti was obtained using pure B. microti DNA samples with serial concentrations and whole blood samples with different densities of B. microti-infected red blood cells. Thereafter, whole mouse blood with a B. microti density lower than that of the nested PCR detection limit and human blood samples infected with B. microti were transfused into healthy mice to assess the transmission risk in mouse models. The infection status of these mice was evaluated through microscopic examination, nested PCR tests, and ELISA. Results: The mice inoculated with different densities of B. microti reached the peak infection rate on different days. Overall, the higher the blood B. microti density was, the earlier the peak infection rate was reached. The levels of specific antibodies against B. microti in the blood of the infected mice increased sharply during the first 30 days of infection, reaching a peak level at 60 days post-infection, and maintaining a high level thereafter. The nested PCR detection limits of B. microti DNA and parasite density were 3 fg and 5.48 parasites/µL, respectively. The whole blood containing an extremely low density of B. microti and human blood samples infected with B. microti could infect mice, confirming the transmission risk of transfusing blood with low-density B. microti. Conclusion: Whole blood containing extremely low density of B. microti poses a high transmission risk when transfused between mice and mice or human and mice, suggesting that Babesia detection should be considered by governments, hospitals, and disease prevention and control centers as a mandatory test before blood donation or transfusion.

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.

Effectiveness of Two New Endochin-like Quinolones, ELQ-596 and ELQ-650, in Experimental Mouse Models of Human Babesiosis.

Endochin-like quinolones (ELQs) define a class of small molecule antimicrobials that target the mitochondrial electron transport chain of various human parasites by inhibiting their cytochrome bc1 complexes. The compounds have shown potent activity against a wide range of protozoan parasites, including the intraerythrocytic parasites Plasmodium and Babesia, the agents of human malaria and babesiosis, respectively. First-generation ELQ compounds were previously found to reduce infection by Babesia microti and Babesia duncani in animal models of human babesiosis but achieved a radical cure only in combination with atovaquone and required further optimization to address pharmacological limitations. Here, we report the identification of two second-generation 3-biaryl ELQ compounds, ELQ-596 and ELQ-650, with potent antibabesial activity in vitro and favorable pharmacological properties. In particular, ELQ-598, a prodrug of ELQ-596, demonstrated high efficacy as an orally administered monotherapy at 10 mg/kg. The compound achieved radical cure in both the chronic model of B. microti-induced babesiosis in immunocompromised mice and the lethal infection model induced by B. duncani in immunocompetent mice. Given its high potency, favorable physicochemical properties, and low toxicity profile, ELQ-596 represents a promising drug for the treatment of human babesiosis.