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

Abstract

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.

Human babesiosis is a rapidly emerging tickborne disease caused by intraerythrocytic Babesia parasites. These parasites are members of the phylum Apicomplexa, which encompasses other pathogens including Plasmodium, Toxoplasma, Cryptosporidium, and Theileria. Similarities in clinical presentation and parasite morphology in babesiosis and malaria [1, 2] can hamper differential diagnosis [3, 4]. The United States Centers for Disease Control and Prevention reported 16,456 clinical cases between 2011 and 2019 in 37 states, 98.2% of which were reported from 10 states [5]. Eight Babesia species have been demonstrated to induce varying degrees of babesiosis, occasionally leading to fatal consequences [2, 6]. Most cases are caused by Babesia microti, with severe cases more frequent in the immunocompromised [2, 6, 7]. Babesia divergens and Babesia duncani can cause fulminant and possibly lethal infections in both immunocompetent and immunocompromised individuals [1, 8, 9]. Recent multiomics studies of B. microti, B. duncani, B. divergens, and Babesia MO1 have unraveled unique mechanisms of survival and adaptation distinct from those of other apicomplexan parasites while also revealing vulnerabilities [10, 11]. Clinical therapies (quinine plus clindamycin or atovaquone plus azithromycin) are effective in many cases, but require large doses and long treatment periods, potentially resulting in mild to severe side effects [12]. Recrudescence following treatment with commonly used therapies has been associated with the emergence of atovaquone- and azithromycin-resistant B. microti, especially among immunocompromised patients [13–16]. Clinical reports of confirmed human babesiosis cases caused by B. duncani in both immunocompetent and immunocompromised patients have described successful treatment with quinine and clindamycin (in most cases 600–650 mg, 3–4 times daily was used) or a combination of atovaquone, proguanil, and azithromycin [17–20]. In four clinical cases from northern California reported by Persing and colleagues [21], 1 of 4 patients had an initial parasitemia of 54% and succumbed to B. duncani infection despite treatment with clindamycin, quinidine, doxycycline, pentamidine, red-cell transfusion, and hemodialysis [21]. In patients with severe babesiosis with parasitemia levels exceeding 10% and associated end-organ dysfunction, red blood cell exchange transfusion is often recommended as a treatment [12, 22]. In vitro and in vivo efficacy studies in mice have confirmed antibabesial activity of atovaquone against B. microti and B. duncani, whereas no curative activity of quinine, clindamycin or azithromycin could be demonstrated at doses up to 50 mg/kg [23, 24]. Emergence of atovaquone-resistant parasites carrying mutations in the Cytb gene encoding a component of the mitochondrial bc₁ complex has been reported [12, 24]. Therapies specific to Babesia parasites and validated through in vitro and in animal models of babesiosis are thus needed to achieve cure with no recrudescence [12].

The 8-aminoquinoline analogue tafenoquine is an approved drug for malaria prophylaxis and the radical cure of Plasmodium vivax [25, 26]. It was initially screened for antibabesial activity in 1997 and since then multiple in vivo studies have demonstrated efficacy against B. microti, Babesia rodhaini, and Plasmodium berghei in mice, and Babesia gibsoni in dogs [27–30]. Tafenoquine has been used to treat 3 immunocompromised patients with human babesiosis. In 1 case, infection with B. microti tolerant to azithromycin and atovaquone was cleared by a 600-mg loading/200-mg weekly dose of tafenoquine [13]. Another case in an immunosuppressed patient with type 2 diabetes and peak parasitemia of over 4% was cured with a similar regimen [31]. However, a third immunocompromised patient infected with B. microti and treated with tafenoquine for 6 weeks was not cured [14]. These data warrant the evaluation of tafenoquine activity in combination with other drugs to enhance their antiparasitic activity while also reducing their toxicity.

Here we demonstrate tafenoquine's efficacy against several Babesia species in vitro and the ability to cure mice from B. microti and B. duncani infections. We also show that tafenoquine plus atovaquone combination cures mice with no recrudescence in a lethal infection model. Finally, we show that tafenoquine-mediated cure leads to sterile immunity to B. duncani.

METHODS

Materials

Tafenoquine (SML0396), atovaquone (A7986), chloroquine (C6628), amodiaquine (1031004), piperaquine (C7874), primaquine (160393), pyrimethamine (SML3579), dihydrorhodamine 123 (D23806), and Hoechst H33342 (H3570) were commercially purchased with >97% purity.

Animal Studies

C3H/HeJ and C.B17/SCID mice were obtained from The Jackson Laboratory and Envigo. Animals were acclimatized for 1 week after arrival before the start of an experiment. Animals that showed signs of distress or appeared moribund were humanly euthanized using approved protocols. All animal experiments followed Yale University institutional guidelines and were approved by the Institutional Animal Care and Use Committee at Yale University (protocol No. 2023-11619). All studies involving the use of human blood and Babesia parasites in culture were approved by the Institutional Biosafety Committee at Yale University.

In Vitro Culture of B. duncani

B. duncani (WA-1 isolate) parasites were cultured in human red blood cells (RBCs) [23, 32, 33]. Established media and conditions were utilized to maintain parasite cultures (Supplementary Methods).

In Vitro Drug Efficacy Studies

In vitro B. duncani parasite drug efficacy experiments were carried out as described previously [34]. The parasites were subjected to serially diluted drugs, and growth assessed after 3 intraerythrocytic life cycles (Supplementary Methods).

Estimation of Reactive Oxygen Species in Tafenoquine-Treated B. duncani Parasites

Cultures of B. duncani-infected RBCs were initiated at 0.5% parasitemia in 2 mL culture volume and treated with tafenoquine (3 µM), atovaquone (0.075 µM), pyrimethamine (0.6 µM), or artemisinin (3 µM) for 62 hours or H₂O₂ (100 µM) for 1 hour. After treatment, cells were washed, resuspended in 1 mL 1× phosphate-buffered saline (PBS), then incubated at 37° C for 30 minutes in the dark with 0.25 µg/mL of dihydrorhodamine 123 (DHR123) and Hoechst H33342, then washed in 1 × PBS and resuspended in 1 mL 1 × PBS. Stained uninfected human RBCs and unstained B. duncani-infected RBCs were used as negative controls. Cells stained with rhodamine and Hoechst were analyzed by flow cytometry (BD LSRII) to measure the fraction of reactive oxygen species (ROS)-positive cells. Data were plotted using GraphPad Prism version 9.1.4. from 3 independent experiments with biological triplicates and represented as mean (±SD).

In Vitro Drug-Drug Interaction

To evaluate the interaction between tafenoquine and atovaquone, the fixed ratio method of in vitro drug combination assay was employed [35]. Parasites were exposed to the 2 drugs in 9 fixed ratios of serially diluted combination of drugs, and their growth was assessed after 3 generations (Supplementary Methods).

In Vivo Drug Efficacy in B. duncani- and B. microti-Infected Mice

The in vivo efficacy studies involving B. duncani and B. microti were conducted in accordance with established animal guidelines and are described in the Supplementary Methods.

Measurement of Antibody Titers by ELISA

A standard indirect enzyme-linked immunosorbent assay (ELISA) was performed to detect total B. duncani parasite antigens, following the procedures described in the Supplementary Methods.

RESULTS

Evidence of Broad-Spectrum Antibabesial Efficacy of Tafenoquine

To assess the efficacy of tafenoquine against Babesia parasites, dose-response assays were conducted in continuous in vitro cultures of B. duncani (WA-1 isolate), B. divergens (Rouen 87 strain), and Babesia MO1 in human erythrocytes. As shown in Figure 1A and Supplementary Table 1, the compound's efficacy in vitro was found to be in the low micromolar range with half maximal inhibitory concentration (IC₅₀) values of approximately 2 µM for B. divergens, approximately 4 µM for B. duncani, and approximately 15 µM for Babesia MO1. A similar range of potency for tafenoquine was determined for Plasmodium falciparum drug-sensitive (3D7) and drug-resistant (Dd2 and HB3) strains (IC₅₀ values, 4 µM for 3D7, 1 µM for Dd2, and 11 µM for HB3; Figure 1A, Supplementary Figure 1, and Supplementary Table 1) consistent with earlier reports [36]. Among 6 quinolines evaluated in this study, tafenoquine showed the highest activity against B. duncani whereas piperaquine was the most active drug against B. divergens with an IC₅₀ of 600 nM (Figure 1B and Supplementary Table 1). Interestingly, Babesia MO1, a close relative of B. divergens (approximately 96% genome-wide nucleotide identity), was unusually tolerant to piperaquine as well as the other quinolines (Figure 1A and 1B, and Supplementary Table 1).

We investigated tafenoquine's ability to alter B. duncani development over 3 consecutive intraerythrocytic life cycles. After 62 hours of growth in the absence of drug treatment, B. duncani developmental forms consisted of mature rings (approximately 39%), tetrads (approximately 25%), early rings (approximately 18%), and filamentous forms (approximately 18%) (Figure 1C). Following treatment with tafenoquine for 62 hours, the mature ring stage was the predominant form (approximately 69%) while filamentous forms and replicating parasites (tetrads) comprised approximately 9% and approximately 6%, respectively (Figure 1C). This suggests that tafenoquine may block Babesia progression in stages with high energy requirements. As a control, treatment with the antifolate pyrimethamine (which inhibits Babesia dihydrofolate reductase thymidylate synthase [DHFR-TS] and consequently purine and pyrimidine biosynthesis) blocked parasite development in the early ring stage (Figure 1C).

Tafenoquine Protects Mice From Lethal B. duncani Infection

Given tafenoquine's potency in vitro against Babesia parasites, we assessed its efficacy in a lethal model of B. duncani infection in immunocompetent C3H/HeJ mice [23, 37]. Tafenoquine (10 mg/kg) was administered daily from 3 to 7 days postinfection (dpi) by oral gavage to mice inoculated with high-dose (HD = 10⁶) or low-dose (LD = 10³) of B. duncani-infected erythrocytes. Vehicle (PEG-400)-treated mice showed rapid parasitemia onset and succumbed to infection either by 6 dpi or 11 dpi following HD and LD inoculation, respectively (Figure 2C and 2D). Tafenoquine-treated mice cleared parasitemia by the last day of treatment (7 dpi) and survived to 45 dpi (Figure 2). Treatment with atovaquone (10 mg/kg) achieved similar results consistent with previous reports (Figure 2) [34]. Parasite clearance following treatment was confirmed by quantitative polymerase chain reaction (qPCR) (Supplementary Figure 2A).

Tafenoquine Eliminates B. microti Infection in Immunocompromised Mice

We next evaluated tafenoquine's in vivo efficacy in a SCID mouse model of B. microti infection [24]. SCID mice inoculated with low (LD = 10⁴) or high (HD = 10⁷) doses of B. microti-infected erythrocytes were treated with tafenoquine or atovaquone at 10 mg/kg daily from 3 to 7 dpi. Vehicle (PEG-400)-treated mice showed a rapid increase in parasitemia starting at 5 dpi or 17 dpi following inoculation with either a high (HD) or a low (LD) infectious inoculum, respectively, after which parasitemia plateaued above 60% (Figure 3). In contrast, in LD mice, tafenoquine treatment prevented parasitemia throughout the entire study (Figure 3A and 3B, and Supplementary Figure 2B). In HD mice, tafenoquine treatment resulted in clearance of infection in 4 of 6 mice whereas the remaining 2 displayed elevated parasitemia at 32 dpi. LD and HD atovaquone-treated mice experienced recrudescence (Figure 3C and 3D), consistent with previous reports [34]. Mutations in the mitochondrial gene Cytb, which encodes the cytochrome b (Cyt-b) subunit of the bc₁ complex, have previously been observed in atovaquone-resistant B. duncani and B. microti parasites collected following treatment [24, 34, 37]. In contrast, we amplified Cytb gene from our recrudescent B. microti parasites and found no such mutations. We further examined the susceptibility to tafenoquine of the recrudescent parasites isolated from the 2 male SCID mice treated with tafenoquine that experienced recrudescence (Figure 3B). Balb/c mice were infected with these isolates and treated with tafenoquine at 10 mg/kg for 5 days starting at 3 dpi. Whereas untreated mice reached 36% parasitemia at 13 dpi, tafenoquine-treated mice were cured (Supplementary Figure 3).

Tafenoquine Is Effective Against Atovaquone-Resistant Babesia Parasites

To assess whether tafenoquine is effective against drug-resistant Babesia parasites, we evaluated its efficacy against B. duncani atovaquone-resistant (BdATV^R) parasites selected in vitro [10]. The BdATV^R strain expresses a Cytb enzyme with a missense amino acid substitution of leucine to phenylalanine residue at position 117 (Figure 4A) [10]. As shown in Figure 4B, the BdATV^R clone is 7 times more resistant to atovaquone than the atovaquone-sensitive (BdATV^S) clone (IC₅₀ approximately 0.167 µM for BdATV^S vs IC₅₀ approximately 1.197 µM for BdATV^R), whereas both clones were equally sensitive to tafenoquine (Figure 4B). Given no indication of cross-resistance in vitro, we then evaluated tafenoquine's ability to cure mice infected with BdATV^R parasites. As shown in Figure 4C, tafenoquine was equally effective in clearing parasitemia and rescuing mice from lethal infection following infection with BdATV^S and BdATV^R parasites. Vehicle-treated groups exhibited parasitemia levels reaching up to 8% and succumbed to infection by 12 dpi (Figure 4D), demonstrating that tafenoquine retains activity against atovaquone-resistant parasites.

Tafenoquine and Atovaquone Increase ROS Production in Babesia Parasites and Their Combination Has Additive Efficacy

While the exact mechanism of action of tafenoquine remains unknown, several studies have highlighted the ability of the compound to cause oxidative stress-related damage due to an increase in the levels of ROS [29]. We quantified the conversion of the nonfluorescent probe DHR123 to its fluorescent product rhodamine 123 in the absence or presence of tafenoquine in B. duncani-infected human RBCs. ROS levels were also measured following treatment with either hydrogen peroxide or artemisinin, both known to induce ROS production [29, 38]. All drugs were dosed at their IC₅₀ concentrations. Cell sorting by fluorescence-activated cell sorting (FACS) was used to measure the fraction of cells accumulating fluorescent rhodamine (ROS positive). Approximately 75% of B. duncani-infected RBCs treated with artemisinin were ROS positive (Figure 5A). Fifty percent of tafenoquine- and atovaquone-treated parasites were ROS positive, whereas pyrimethamine-treated parasites showed baseline ROS levels at approximately 6% (Figure 5A). Fluorescence microscopy further confirmed the FACS data (Figure 5B). These data indicate that tafenoquine and atovaquone cause oxidative damage. This finding led us to investigate the impact of a combination of tafenoquine and atovaquone on B. duncani in culture. As shown in Figure 5C, tafenoquine and atovaquone interact additively with the sum of the fractional 50% inhibitory concentration (ƩFIC₅₀) of 1.04. These data suggest that tafenoquine plus atovaquone could be a viable antibabesial combination therapy to eliminate drug-resistant parasites.

A Combination of Tafenoquine and Atovaquone Eliminates Babesia Infection With no Recrudescence

The additive effect of tafenoquine and atovaquone led us to investigate the efficacy of this combination in vivo in B. duncani and B. microti mouse models. Rewrite the sentence as follows: C3H/HeJ mice (n = 6 per group) infected with LD = 10³ or HD = 10⁶B. duncani-infected RBCs were treated with either vehicle or tafenoquine (10 mg/kg) and atovaquone (10 mg/kg) once a day for 5 days starting at day 3 post-infection. Vehicle-treated LD mice developed 4% parasitemia and were euthanized by 12 dpi, while vehicle-treated HD mice reached high parasitemia levels (up to 25%), and were euthanized by 6 dpi (Figure 6A and 6B). Tafenoquine+atovaquone-treated groups cleared infection with no detectable parasitemia and survived through 45 dpi (Figure 6B). CB17/SCID mice infected with B. microti-infected erythrocytes treated with tafenoquine plus atovaquone showed no signs of parasitemia through 45 dpi (Figure 6C), whereas vehicle-treated LD and HD mice reached high parasitemia by 20 dpi and 7 dpi, respectively (Figure 6C). In contrast, mice infected with B. microti and treated with atovaquone all displayed a recurrence of parasitemia (Figure 3C and 3D). Drug combinations consisting of atovaquone plus azithromycin or clindamycin plus quinine showed little to no efficacy in mice (see summary in Supplementary Table 2).

Tafenoquine Treatment Confers Sterile Immunity and Protects Mice From Lethal B. duncani Challenge

We next investigated whether mice cured following treatment with tafenoquine could be protected from subsequent Babesia infections. C3H/HeJ mice (n = 6) were treated with tafenoquine (10 mg/kg, 1–10 dpi) to clear parasitemia after infection with a lethal dose of 10⁶B. duncani-infected RBCs then challenged again at 34 dpi. All tafenoquine-cured mice showed no detectable parasitemia and survived, as did the tafenoquine plus atovaquone-treated mice (Figure 7A and 7C, and Supplementary Figure 4A and 4C). In contrast, vehicle-treated and age-matched untreated control mice succumbed to infection by 6 dpi with parasitemia up to 35% following challenge (Figure 7A and 7C, and Supplementary Figure 4A and 4C). qPCR detected parasite DNA during drug treatment on 6 dpi, whereas no detectable parasite DNA could be detected on 28 dpi during both the treatment and challenge phases (Figure 7B and Supplementary Figure 4B). Consistent with the apparent immune protection, antibodies to B. duncani antigens increased during the treatment phase and again during the challenge phase (Figure 7D).

DISCUSSION

In this study, we demonstrated the effectiveness of the antimalarial 8-aminoquinoline tafenoquine against B. duncani (WA-1 isolate), B. divergens (Rouen 87 strain), and Babesia MO1 in vitro, and B. duncani and B. microti in mice. These findings add to tafenoquine's earlier successes against other protozoan parasites including P. falciparum, P. vivax, Trypanosoma brucei, and Toxoplasma gondii as well as nonhuman parasites such as B. bovis, B. rodhaini (rodent) and B. gibsoni (canine) [28, 29, 39, 40]. Tafenoquine has been under development since late 1970s, and in 2018 received approval from the US Food and Drug Administration as an antimalarial, making it the second 8-aminoquinoline after primaquine to reach the clinic [41]. Tafenoquine has been shown to be as effective as other quinolines at treating P. vivax infections, including primaquine, but its longer half-life (Supplementary Table 3) allows a shorter time course, enhancing patient compliance and reducing cost [42]. Considering its contraindication in glucose-6-phosphate dehydrogenase (G6PD)-deficient patients [42] and mixed results in off-label use as a monotherapy against babesiosis, further preclinical and clinical evaluations of tafenoquine alone or in combination with other antiparasitic drugs are needed.

Our studies showed that tafenoquine monotherapy at a dose of 10 mg/kg administered daily for 5 days to mice infected with B. duncani resulted in elimination of parasitemia and survival of the infected mice from an otherwise lethal infection, whereas 4 out of 6 mice infected with B. microti cleared the parasite in SCID mice. It is noteworthy that the 10 mg/kg dose of tafenoquine in mice correspond to approximately 20% the calculated human equivalent dose (40–60 mg/kg) (Supplementary Tables 3 and 4). Therefore, higher doses of tafenoquine monotherapy could possibly also clear infection. In support of this hypothesis, a previous study on the efficacy of tafenoquine monotherapy against B. microti in Syrian hamsters using a dose of 52 mg/kg for 4 days showed elimination of parasitemia in animals [21] (Supplementary Table 4). Interestingly, the recrudescent B. microti parasites isolated from the SCID mice following treatment with tafenoquine monotherapy at 10 mg/kg for 5 days were found to be sensitive to tafenoquine upon reinoculation, highlighting the need of further investigation of the mechanism of recrudescence by Babesia parasites. Of clinical relevance, a recent report showed failure of tafenoquine monotherapy in an immunocompromised patient despite a 46-day course (loading dose of 200 mg daily for 3 days, then 200 mg once per week for 43 days) [14]. Together, these findings suggest that a combination of tafenoquine with another potent antibabesial drug could be beneficial in achieving radical cure even among immunocompromised individuals.

Combinations of tafenoquine with various drugs, particularly chloroquine, have previously been evaluated for human malaria. However, a combination of tafenoquine and chloroquine is not suitable for human babesiosis as chloroquine has little to no efficacy against Babesia parasites [12, 43]. In this study, we found that a combination of tafenoquine and atovaquone achieves radical cure in both immunocompromised and immunocompetent mice infected with B. microti or B. duncani, respectively. Our study has focused primarily on a combination of tafenoquine and atovaquone. Other studies aimed at determining the efficacy of tafenoquine in mice with other partner drugs, such as those approved for the treatment of human babesiosis (azithromycin and clindamycin) or other antiparasitic drugs as was shown for artesunate in mice [28], are thus warranted.

Although the exact mechanism of action of tafenoquine remains poorly understood, the drug is thought to act against malaria parasites via oxidative stress brought on by drug-induced mitochondrial dysfunction and/or inhibition of receptor recycling by endosomes [44]. Our discovery that tafenoquine arrests Babesia parasite development in the most metabolically active stage, and our observation that it causes ROS production, suggest that it imparts oxidative stress, consistent with earlier reports that tafenoquine disrupts mitochondrial function in parasites [45]. Atovaquone is also known to disrupt mitochondrial function through inhibition of the bc₁ complex [24], and as shown in our study also results in an increase in ROS levels in B. duncani (Figure 4B). Our data also showed that tafenoquine is effective against an atovaquone-resistant B. duncani clone in vitro and in vivo, with additive synergy in vitro. Altogether, it seems reasonable to conclude that tafenoquine and atovaquone both disrupt parasite mitochondria but in distinct ways, making them excellent partner drugs.

While this study found that tafenoquine and atovaquone outperformed other combinations including atovaquone and azithromycin in our mouse model, further superiority testing will be needed to make the case for clinical translation of this novel combination. Another important factor to consider when evaluating the potential for translation of combinations including tafenoquine is the severe hemolytic anemia tafenoquine and other 8-aminoquinolines (including primaquine) cause in humans with G6PD deficiency. Veterinary applications in dogs and cattle (in which species G6PD incidence is far lower than in humans) may represent an avenue of accelerated translation potential.

Beyond clearing infection, tafenoquine alone or in combination with atovaquone confers orthologous sterile immunity. Unfortunately, in human babesiosis the standard treatment regime is often associated with relapse, especially in the context of treatment with immunosuppressives such as rituximab. Immunization via controlled infection has been extensively researched in rodents, nonhuman primates, and humans using sporozoites, and entails initiation of an infection that is subsequently controlled by drugs, resulting in strong protection following homologous challenge [46–48]. However, it has been observed that this approach offers little to no blood-stage protection in humans [49]. We hypothesize that parasite killing by tafenoquine alone or in combination with atovaquone leads to the release of parasite proteins, which serve as antigens that trigger a robust immune response. This hypothesis is supported by our findings of elevated antibodies against secreted Babesia antigens in sera from treated mice. Drug-mediated immune protection has the potential to shed considerable light on the challenging aspects of vaccine development for human babesiosis.

In conclusion, our studies using experimental models of human babesiosis identified that treatment with a combination of tafenoquine and atovaquone is a highly promising therapy to achieve radical cure as well as provide immune protection to immunocompetent Babesia-infected patients from subsequent infections. The success of this therapeutic strategy for the treatment of human babesiosis could pave the way for its use in the treatment of infections caused by other apicomplexan parasites.

Supplementary Data

Supplementary materials are available at The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.

Notes

Author contributions. P. V. contributed investigation, methodology, analysis, visualization, writing the original draft, review, and editing. A. C. P. contributed investigation, methodology, review, and editing. I. R. and V. K. contributed investigation and methodology. M. C. contributed investigation, visualization, and methodology. J. C. G. contributed analysis, review, and editing. C. B. M. contributed conceptualization, supervision, funding acquisition, project administration, writing original draft, review, and editing. All authors have read and agreed the published version of the manuscript.

Financial support. This work was supported by the National Institutes of Health (grant numbers AI123321, AI138139, AI152220, and AI136118); the Steven and Alexandra Cohen Foundation (grant number Lyme 62 2020); and the Global Lyme Alliance.

References

Author notes