Evaluation on the in vivo efficacy of malarone and atovaquone-azithromycin combination against Babesia microti in mice under different immune status

doi:10.12140/j.issn.1000-7423.2021.05.014

Abstract

Abstract:

Objective To evaluate the in vivo efficacy against Babesia microti of two therapies commonly used in the clinic: a combination of atovaquone (ATQ) with azithromycin (AZM) and malarone in immune-normal BALB/c and non-obese diabetic/severe combined immunodeficient(NOD/SCID) mice models. Methods In total, 69 BALB/c and 15 NOD/SCID mice were each inoculated with 10⁷ Babesia microti-infected erythrocytes. The mice of each immune type were divided into three groups: ATQ + AZM group (195 mg/kg ATQ + 32.5 mg/kg AZM), malarone group (62.5 mg/kg ATQ + 25 mg/kg proguanil), and control group (5% soluble starch solution), the drug was administered by gavage of 0.2 ml/10 g body weight. In the drug-suppression testing in BALB/c mice(12 mice each of two drug groups, and 15 mice in the control group), the mice received the first dose of drug 4 h after infection, and then the dosing was continued for 10 consecutive days. Three mice were randomly selected for blood sampling from tail tip before gavage administration on 1, 3, 5, 7, and 10 days post-infection. Thin blood smears were prepared for microscopy to examine B. microti infection of erythrocytes, and estimate the erythrocyte infection rate (EIR); the blood samples were tested for 18S rRNA using qPCR to examine the gene value. In the drug-therapy testing experiment with BALB/c mice (10 mice each of 3 group), tail tip blood samples were collected from all mice 7 d post-infection to determine whether the infection was established, then when the infection was confirmed, the drug administration was initiated and continued for 10 consecutive days; microscopic examination was conducted for the blood samples collected on days 10, 11, 12, 13, 15, 17, 19, 24 and 27 post-infection to estimate the EIR. On 27 d post-infection, 5 mice randomly selected from each group were intraperitoneally injected with immunosuppressant dexamethasone sodium phosphate solution (200 μl/mouse) for 7 consecutive days; starting on 3 d after immunosuppressant injection, daily blood samples were examined by staining microscopy to observe recrudescence; Orbital sinus blood samples were collected from the mice randomly selected (5 from each group), and the anticoagulated whole blood samples obtained were pooled and injected intraperitoneally to corresponding groups of normal BALB/c (5 mice/group); on 7-10 d post subinnoculation, blood samples were examined to estimate EIR. In the experiment with NOD/SCID mice (3 groups, 5 mice each group), drugs were administered from 10 d post infection and continued for 10 days. Tail tip blood samples of the mice of three groups were collected on 10, 12, 15, 17, 19, 21, 24, 27, 29, 31, 35, 42 and 49 d post-infection, and examined by staining microscopy to estimate EIR. Results In the drug-suppression test in BALB/c mice, both ATQ + ATM and malarone obviously suppressed parasitemia. Microscopic examination indicated the only one mouse in the ATQ + AZM group was found infected on 3 d and 5 d post infection, with EIR of (0.20 ± 0.12)% and (0.30 ± 0.17)% respectively, while decreased to 0 on 7 d post-infection (8 d post-dosing). EIR was 0 in all mice in the malarone group. Significant differences were observed in EIR of control vs ATQ + ATM group and control vs malarone group (F = 151.6, 153.5, P < 0.05). The relative value of 18S rRNA gene was at 0.010 2 ± 0.001 2 and 0.007 8 ± 0.006 6 in malarone and the ATQ + AZM groups, respectively on 7 d post infection, which was significantly different from the control group (68.143 8 ± 79.122 9) (F = 7.376, 7.382, P < 0.05). On 10 d post-infection (1 d upon drug withdrawal), the value of 18S rRNA gene decreased to 0.001 7 ± 0.000 9 and 0.000 8 ± 0.000 6 in the malarone and ATQ + AZM groups on day 10 post-infection, respectively, which were significantly different from the control group (t = 4.229, 4.229, P < 0.05). In the drug-therapy tests with BALB/c, EIR in control, ATQ + AZM, and malarone groups peaked on 7 d post-infection, (36.67 ± 10.85)%, (35.30 ± 6.46)%, and (33.53 ± 7.37)% respectively, and decreased to(10.47 ± 8.02)%, (1.53 ± 0.31)% and (6.27 ± 1.01)%, respectively, on 11 d post-infection (5 d upon drug withdrawal); EIR in all groups declined to 0 on 15 d post-infection. B. microti was observed in one mouse in the control group 3 d after immunosuppressant injection, and seen in both the ATQ + AZM and malarone groups 5 d after immunosuppressing, indicating that recrudescence occurred in all groups. Subinoculation experiment showed that three mice that received blood from either the ATQ + AZM or malarone groups developed parasitemia 7 d after subinoculation. One mouse and two mice showed parasitemia in the ATQ + AZM and malarone groups, respectively, 9 d after subinoculation, and no parasite was observed in any mice from 10 d post subinoculation. In the test with NOD/SCID mice, the EIR of ATQ + AZM and malarone groups rapidly reduced after treatment from its peak value on 10 d post infection [(59.90 ± 0.10) % and (59.37 ± 0.35)%, respectively] to 0 on 24 d post-infection, whereas B. microti was observed again on day 29 and 42 post infection. EIR in the control group fluctuated between (47.20 ± 0.80)% and (66.80 ± 0.80)%, and all mice died on 45 d post-infection, of which the process was significantly different from ATQ + ATM, and malarone group (F = 5 505 and 5 984, respectively, P < 0.05). Conclusion Both ATQ + ATM and malarone that commonly used in clinic showed suppressive effect to some extent on the proliferation of B. microti in infected mice, but could not clear up all the parasites; the blood of mice remains infective after drug treatment and recrudescence may occur in immunocompromised hosts, causing high infection rate of erythrocyte.

Key words: Babesia microti, Atovaquone, Azithromycin, Malarone, Drug efficacy evaluation, Erythrocyte infected rate

CLC Number:

R531.9

YIN Meng, ZHANG Hao-bing, TAO Yi, JIANG Bin, LIU Hua. Evaluation on the in vivo efficacy of malarone and atovaquone-azithromycin combination against Babesia microti in mice under different immune status[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2021, 39(5): 659-665.

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References 28

[1]	Vannier E, Krause PJ. Human babesiosis[J]. N Engl J Med, 2012, 366(25):2397-2407.
[2]	Vannier EG, Diuk-Wasser MA, Ben Mamoun C, et al. Babesiosis[J]. Infect Dis Clin N Am, 2015, 29(2):357-370.
[3]	Ruebush TK. Human babesiosis in north America[J]. Trans R Soc Trop Med Hyg, 1980, 74(2):149-152.
[4]	Patel KM, Johnson JE, Reece R, et al. Babesiosis-associated splenic rupture: case series from a hyperendemic region[J]. Clin Infect Dis, 2019, 69(7):1212-1217.
[5]	Gray EB, Herwaldt BL. Babesiosis surveillance--United States, 2011—2015[J]. MMWR Surveill Summ, 2019, 68(6):1-11.
[6]	Goo YK, Terkawi MA, Jia H, et al. Artesunate, a potential drug for treatment of Babesia infection[J]. Parasitol Int, 2010, 59(3):481-486.
[7]	Lawres LA, Garg A, Kumar V, et al. Radical cure of experimental babesiosis in immunodeficient mice using a combination of an endochin-like quinolone and atovaquone[J]. J Exp Med, 2016, 213(7):1307-1318.
[8]	El-Sayed SAES, Rizk MA, Ringo AE, et al. Impact of using pyronaridine tetraphosphate-based combination therapy in the treatment of babesiosis caused by Babesia bovis, B. caballi, and B. gibsoni in vitro and B. microti in mice[J]. Parasitol Int, 2021, 81:102260.
[9]	Marley SE, Eberhard ML, Steurer FJ, et al. Evaluation of selected antiprotozoal drugs in the Babesia microti-hamster model[J]. Antimicrob Agents Chemother, 1997, 41(1):91-94.
[10]	Wormser GP, Dattwyler RJ, Shapiro ED, et al. The clinical assessment, treatment, and prevention of Lyme disease, human granulocytic anaplasmosis, and babesiosis: clinical practice guidelines by the Infectious Diseases Society of America[J]. Clin Infect Dis, 2006, 43(9):1089-1134.
[11]	Vial HJ, Gorenflot A. Chemotherapy against babesiosis[J]. Vet Parasitol, 2006, 138(1/2):147-160.
[12]	Sanchez E, Vannier E, Wormser GP, et al. Diagnosis, treatment, and prevention of Lyme disease, human granulocytic anaplasmosis, and babesiosis: a review[J]. JAMA, 2016, 315(16):1767-1777.
[13]	Krause PJ, Gewurz BE, Hill D, et al. Persistent and relapsing babesiosis in immunocompromised patients[J]. Clin Infect Dis, 2008, 46(3):370-376.
[14]	Wormser GP, Prasad A, Neuhaus E, et al. Emergence of resistance to azithromycin-atovaquone in immunocompromised patients with Babesia microti infection[J]. Clin Infect Dis, 2010, 50(3):381-386.
[15]	Guirao-Arrabal E, González LM, García-Fogeda JL, et al. Imported babesiosis caused by Babesia microti--a case report[J]. Ticks Tick Borne Dis, 2020, 11(4):101435.
[16]	Vyas JM, Telford SR, Robbins GK. Treatment of refractory Babesia microti infection with atovaquone-proguanil in an HIV-infected patient: case report[J]. Clin Infect Dis, 2007, 45(12):1588-1590.
[17]	Blanshard A, Hine P. Atovaquone-proguanil for treating uncomplicated Plasmodium falciparum malaria[J]. Cochrane Database Syst Rev, 2021, 1: CD004529.
[18]	Xu SY, Bian RL, Chen X. Pharmacological experiment methodology[M]. 3 ed. Beijing: People’s Medical Publishing House, 2002: 1597-1600. (in Chinese)
[18]	( 徐叔云, 卞如濂, 陈修. 药理实验方法学[M]. 3版. 北京: 人民卫生出版社, 2002: 1597-1600.)
[19]	Zhao W, Sun GZ. Dose conversion between different kinds of experimental animals[J]. Chin J Animal Husb Vet Med, 2010(5):52-53. (in Chinese)
[19]	( 赵伟, 孙国志. 不同种实验动物间用药量换算[J]. 畜牧兽医科技信息, 2010(5):52-53.)
[20]	Teal AE, Habura A, Ennis J, et al. A new real-time PCR assay for improved detection of the parasite Babesia microti[J]. J Clin Microbiol, 2012, 50(3):903-908.
[21]	Beshbishy AM, Batiha GE, Yokoyama N, et al. Ellagic acid microspheres restrict the growth of Babesia and Theileria in vitro and Babesia microti in vivo[J]. Parasit Vectors, 2019, 12(1):269.
[22]	Yao JM, Zhang HB, Liu CS, et al. Inhibitory effects of 19 antiprotozoal drugs and antibiotics on Babesia microti infection in BALB/c mice[J]. J Infect Dev Ctries, 2015, 9(9):1004-1010.
[23]	Mordue DG, Wormser GP. Could the drug tafenoquine revolutionize treatment of Babesia microti infection?[J]. J Infect Dis, 2019, 220(3):442-447.
[24]	Tuvshintulga B, Vannier E, Tayebwa DS, et al. Clofazimine, a promising drug for the treatment of Babesia microti infection in severely immunocompromised hosts[J]. J Infect Dis, 2020, 222(6):1027-1036.
[25]	Nehrbass-Stuedli A, Boykin D, Tidwell RR, et al. Novel diamidines with activity against Babesia divergens in vitro and Babesia microti in vivo[J]. Antimicrob Agents Chemother, 2011, 55(7):3439-3445.
[26]	Wang S, Li M, Luo X, et al. Inhibitory effects of fosmidomycin against Babesia microti in vitro[J]. Front Cell Dev Biol, 2020, 8:247.
[27]	Guo JY, Luo XY, Wang S, et al. Xanthohumol and gossypol are promising inhibitors against Babesia microti by in vitro culture via high-throughput screening of 133 natural products[J]. Vaccines, 2020, 8(4):613.
[28]	AbouLaila M, Sivakumar T, Yokoyama N, et al. Inhibitory effect of terpene nerolidol on the growth of Babesia parasites[J]. Parasitol Int, 2010, 59(2):278-282.

免疫抑制后天数/d Days post-immunosuppressant injection/d	对照组 Control group	ATQ + AZM组 ATQ + AZM group	马拉龙组 Malarone group
3	0.06 ± 0.13	0	0
5	0.25 ± 0.07	0.10 ± 0.14	0.10 ± 0.14
7	1.56 ± 1.31	0.52 ± 0.46	3.20 ± 2.58
9^a	3.04 ± 5.15	0.20 ± 0.28	0.33 ± 0.38

继代接种感染后天数/d Days post subpassage/d	对照组 Control group	ATQ + AZM组 ATQ + AZM group	马拉龙组 Malarone group
7	3.34 ± 1.44	0.12 ± 0.11	0.42 ± 0.41
9	3.12 ± 2.72	0.04 ± 0.09	0.10 ± 0.12
12	32.60 ± 13.99	0	0