Trypanosoma brucei infection protects mice against malaria

English version

Autoři: Margarida Sanches-Vaz ^aff001; Adriana Temporão ^aff001; Rafael Luis ^aff001; Helena Nunes-Cabaço ^aff001; António M. Mendes ^aff001; Sarah Goellner ^aff001; Tânia Carvalho ^aff001; Luisa M. Figueiredo ^aff001; Miguel Prudêncio ^aff001
Působiště autorů: Instituto de Medicina Molecular João Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal ^aff001
Vyšlo v časopise: Trypanosoma brucei infection protects mice against malaria. PLoS Pathog 15(11): e32767. doi:10.1371/journal.ppat.1008145
Kategorie: Research Article
doi: https://doi.org/10.1371/journal.ppat.1008145

Souhrn

Sleeping sickness and malaria are parasitic diseases with overlapping geographical distributions in sub-Saharan Africa. We hypothesized that the immune response elicited by an infection with Trypanosoma brucei, the etiological agent of sleeping sickness, would inhibit a subsequent infection by Plasmodium, the malaria parasite, decreasing the severity of its associated pathology. To investigate this, we established a new co-infection model in which mice were initially infected with T. brucei, followed by administration of P. berghei sporozoites. We observed that a primary infection by T. brucei significantly attenuates a subsequent infection by the malaria parasite, protecting mice from experimental cerebral malaria and prolonging host survival. We further observed that an ongoing T. brucei infection leads to an accumulation of lymphocyte-derived IFN-γ in the liver, limiting the establishment of a subsequent hepatic infection by P. berghei sporozoites. Thus, we identified a novel host-mediated interaction between two parasitic infections, which may be epidemiologically relevant in regions of Trypanosoma/Plasmodium co-endemicity.

Klíčová slova:

Malaria – Malarial parasites – Parasitemia – Parasitic diseases – Plasmodium – Sporozoites – Trypanosoma – Trypanosoma brucei gambiense

Zdroje

1. Cox FE. Concomitant infections, parasites and immune responses. Parasitology. 2001;122 Suppl:S23–38. Epub 2001/07/10. doi: 10.1017/s003118200001698x 11442193.

2. Griffiths EC, Pedersen AB, Fenton A, Petchey OL. The nature and consequences of coinfection in humans. J Infect. 2011;63(3):200–6. Epub 2011/06/28. doi: 10.1016/j.jinf.2011.06.005 21704071; PubMed Central PMCID: PMC3430964.

3. Salgame P, Yap GS, Gause WC. Effect of helminth-induced immunity on infections with microbial pathogens. Nat Immunol. 2013;14(11):1118–26. Epub 2013/10/23. doi: 10.1038/ni.2736 24145791; PubMed Central PMCID: PMC4955540.

4. Salam N, Mustafa S, Hafiz A, Chaudhary AA, Deeba F, Parveen S. Global prevalence and distribution of coinfection of malaria, dengue and chikungunya: a systematic review. BMC Public Health. 2018;18(1):710. Epub 2018/06/09. doi: 10.1186/s12889-018-5626-z 29879935; PubMed Central PMCID: PMC5992662.

5. Kwenti TE. Malaria and HIV coinfection in sub-Saharan Africa: prevalence, impact, and treatment strategies. Res Rep Trop Med. 2018;9:123–36. Epub 2018/08/14. doi: 10.2147/RRTM.S154501 30100779; PubMed Central PMCID: PMC6067790.

6. Hotez PJ, Molyneux DH. Tropical anemia: one of Africa's great killers and a rationale for linking malaria and neglected tropical disease control to achieve a common goal. PLoS Negl Trop Dis. 2008;2(7):e270. Epub 2008/07/31. doi: 10.1371/journal.pntd.0000270 18665256; PubMed Central PMCID: PMC2474697.

7. Prudencio M, Rodriguez A, Mota MM. The silent path to thousands of merozoites: the Plasmodium liver stage. Nat Rev Microbiol. 2006;4(11):849–56. Epub 2006/10/17. doi: 10.1038/nrmicro1529 17041632.

8. Trindade S, Rijo-Ferreira F, Carvalho T, Pinto-Neves D, Guegan F, Aresta-Branco F, et al. Trypanosoma brucei Parasites Occupy and Functionally Adapt to the Adipose Tissue in Mice. Cell Host Microbe. 2016;19(6):837–48. Epub 2016/05/31. doi: 10.1016/j.chom.2016.05.002 27237364; PubMed Central PMCID: PMC4906371.

9. Capewell P, Cren-Travaille C, Marchesi F, Johnston P, Clucas C, Benson RA, et al. The skin is a significant but overlooked anatomical reservoir for vector-borne African trypanosomes. Elife. 2016;5. Epub 2016/09/23. doi: 10.7554/eLife.17716 27653219; PubMed Central PMCID: PMC5065312.

10. Mansfield JM, Paulnock DM. Regulation of innate and acquired immunity in African trypanosomiasis. Parasite Immunol. 2005;27(10–11):361–71. Epub 2005/09/24. doi: 10.1111/j.1365-3024.2005.00791.x 16179030.

11. Ponte-Sucre A. An Overview of Trypanosoma brucei Infections: An Intense Host-Parasite Interaction. Front Microbiol. 2016;7:2126. Epub 2017/01/14. doi: 10.3389/fmicb.2016.02126 28082973; PubMed Central PMCID: PMC5183608.

12. Blum J, Schmid C, Burri C. Clinical aspects of 2541 patients with second stage human African trypanosomiasis. Acta Trop. 2006;97(1):55–64. Epub 2005/09/15. doi: 10.1016/j.actatropica.2005.08.001 16157286.

13. Priotto G, Pinoges L, Fursa IB, Burke B, Nicolay N, Grillet G, et al. Safety and effectiveness of first line eflornithine for Trypanosoma brucei gambiense sleeping sickness in Sudan: cohort study. BMJ. 2008;336(7646):705–8. Epub 2008/03/07. doi: 10.1136/bmj.39485.592674.BE 18321960; PubMed Central PMCID: PMC2276259.

14. MacLean LM, Odiit M, Chisi JE, Kennedy PG, Sternberg JM. Focus-specific clinical profiles in human African Trypanosomiasis caused by Trypanosoma brucei rhodesiense. PLoS Negl Trop Dis. 2010;4(12):e906. Epub 2010/12/15. doi: 10.1371/journal.pntd.0000906 21151878; PubMed Central PMCID: PMC2998431.

15. Kagira JM, Maina N, Njenga J, Karanja SM, Karori SM, Ngotho JM. Prevalence and types of coinfections in sleeping sickness patients in kenya (2000/2009). J Trop Med. 2011;2011:248914. Epub 2011/09/15. doi: 10.1155/2011/248914 21915184; PubMed Central PMCID: PMC3170889.

16. Kuepfer I, Hhary EP, Allan M, Edielu A, Burri C, Blum JA. Clinical presentation of T.b. rhodesiense sleeping sickness in second stage patients from Tanzania and Uganda. PLoS Negl Trop Dis. 2011;5(3):e968. Epub 2011/03/17. doi: 10.1371/journal.pntd.0000968 21407802; PubMed Central PMCID: PMC3046969.

17. Gillet P, Mumba Ngoyi D, Lukuka A, Kande V, Atua B, van Griensven J, et al. False positivity of non-targeted infections in malaria rapid diagnostic tests: the case of human african trypanosomiasis. PLoS Negl Trop Dis. 2013;7(4):e2180. Epub 2013/05/03. doi: 10.1371/journal.pntd.0002180 23638201; PubMed Central PMCID: PMC3636101.

18. Kato CD, Nanteza A, Mugasa C, Edyelu A, Matovu E, Alibu VP. Clinical profiles, disease outcome and co-morbidities among T. b. rhodesiense sleeping sickness patients in Uganda. PLoS One. 2015;10(2):e0118370. Epub 2015/02/27. doi: 10.1371/journal.pone.0118370 25719539; PubMed Central PMCID: PMC4342333.

19. Maina NW, Kagira JM, Oberle M, Ndung'u JJ, Brun R. Co-infection of sleeping sickness patients with malaria and loiasis in southern Sudan. Journal of Protozoology Research. 2010;20(2):12–9.

20. Millott SM, Cox FE. Interactions between Trypanosoma brucei and Babesia spp. and Plasmodium spp. in mice. Parasitology. 1985;90 (Pt 2):241–54. doi: 10.1017/s0031182000050952 4000702.

21. Ademola IO, Odeniran PO. Co-infection with Plasmodium berghei and Trypanosoma brucei increases severity of malaria and trypanosomiasis in mice. Acta Trop. 2016;159:29–35. doi: 10.1016/j.actatropica.2016.03.030 27021269.

22. Ferreira A, Schofield L, Enea V, Schellekens H, van der Meide P, Collins WE, et al. Inhibition of development of exoerythrocytic forms of malaria parasites by gamma-interferon. Science. 1986;232(4752):881–4. doi: 10.1126/science.3085218 3085218.

23. Mellouk S, Green SJ, Nacy CA, Hoffman SL. IFN-gamma inhibits development of Plasmodium berghei exoerythrocytic stages in hepatocytes by an L-arginine-dependent effector mechanism. J Immunol. 1991;146(11):3971–6. 1903415.

24. Vergara U, Ferreira A, Schellekens H, Nussenzweig V. Mechanism of escape of exoerythrocytic forms (EEF) of malaria parasites from the inhibitory effects of interferon-gamma. J Immunol. 1987;138(12):4447–9. 3108391.

25. Prudencio M, Mota MM, Mendes AM. A toolbox to study liver stage malaria. Trends Parasitol. 2011;27(12):565–74. doi: 10.1016/j.pt.2011.09.004 22015112.

26. Vaughan JA, Scheller LF, Wirtz RA, Azad AF. Infectivity of Plasmodium berghei sporozoites delivered by intravenous inoculation versus mosquito bite: implications for sporozoite vaccine trials. Infect Immun. 1999;67(8):4285–9. 10417207; PubMed Central PMCID: PMC96740.

27. Scheller LF, Wirtz RA, Azad AF. Susceptibility of different strains of mice to hepatic infection with Plasmodium berghei. Infect Immun. 1994;62(11):4844–7. 7927764; PubMed Central PMCID: PMC303196.

28. Wykes MN, Good MF. What have we learnt from mouse models for the study of malaria? Eur J Immunol. 2009;39(8):2004–7. doi: 10.1002/eji.200939552 19672886.

29. Zuzarte-Luis V, Sales-Dias J, Mota MM. Simple, sensitive and quantitative bioluminescence assay for determination of malaria pre-patent period. Malar J. 2014;13:15. doi: 10.1186/1475-2875-13-15 24400642; PubMed Central PMCID: PMC3893453.

30. Sa ECC, Nyboer B, Heiss K, Sanches-Vaz M, Fontinha D, Wiedtke E, et al. Plasmodium berghei EXP-1 interacts with host Apolipoprotein H during Plasmodium liver-stage development. Proc Natl Acad Sci U S A. 2017;114(7):E1138–E47. doi: 10.1073/pnas.1606419114 28137845; PubMed Central PMCID: PMC5320984.

31. Pfeil J, Sepp KJ, Heiss K, Meister M, Mueller AK, Borrmann S. Protection against malaria by immunization with non-attenuated sporozoites under single-dose piperaquine-tetraphosphate chemoprophylaxis. Vaccine. 2014;32(45):6005–11. doi: 10.1016/j.vaccine.2014.07.112 25203450.

32. Lewis MD, Behrends J, Sa ECC, Mendes AM, Lasitschka F, Sattler JM, et al. Chemical attenuation of Plasmodium in the liver modulates severe malaria disease progression. J Immunol. 2015;194(10):4860–70. doi: 10.4049/jimmunol.1400863 25862814.

33. Friesen J, Silvie O, Putrianti ED, Hafalla JC, Matuschewski K, Borrmann S. Natural immunization against malaria: causal prophylaxis with antibiotics. Sci Transl Med. 2010;2(40):40ra9. doi: 10.1126/scitranslmed.3001058 20630856.

34. Tavares J, Formaglio P, Thiberge S, Mordelet E, Van Rooijen N, Medvinsky A, et al. Role of host cell traversal by the malaria sporozoite during liver infection. J Exp Med. 2013;210(5):905–15. doi: 10.1084/jem.20121130 23610126; PubMed Central PMCID: PMC3646492.

35. Stienstra R, Saudale F, Duval C, Keshtkar S, Groener JE, van Rooijen N, et al. Kupffer cells promote hepatic steatosis via interleukin-1beta-dependent suppression of peroxisome proliferator-activated receptor alpha activity. Hepatology. 2010;51(2):511–22. doi: 10.1002/hep.23337 20054868.

36. Deshmane SL, Kremlev S, Amini S, Sawaya BE. Monocyte chemoattractant protein-1 (MCP-1): an overview. J Interferon Cytokine Res. 2009;29(6):313–26. doi: 10.1089/jir.2008.0027 19441883; PubMed Central PMCID: PMC2755091.

37. Namangala B, Noel W, De Baetselier P, Brys L, Beschin A. Relative contribution of interferon-gamma and interleukin-10 to resistance to murine African trypanosomosis. J Infect Dis. 2001;183(12):1794–800. doi: 10.1086/320731 11372033.

38. Cnops J, De Trez C, Stijlemans B, Keirsse J, Kauffmann F, Barkhuizen M, et al. NK-, NKT- and CD8-Derived IFNgamma Drives Myeloid Cell Activation and Erythrophagocytosis, Resulting in Trypanosomosis-Associated Acute Anemia. PLoS Pathog. 2015;11(6):e1004964. doi: 10.1371/journal.ppat.1004964 26070118; PubMed Central PMCID: PMC4466398.

39. Schofield L, Ferreira A, Altszuler R, Nussenzweig V, Nussenzweig RS. Interferon-gamma inhibits the intrahepatocytic development of malaria parasites in vitro. J Immunol. 1987;139(6):2020–5. 2957445.

40. Hofstra CL, Van Ark I, Hofman G, Nijkamp FP, Jardieu PM, Van Oosterhout AJ. Differential effects of endogenous and exogenous interferon-gamma on immunoglobulin E, cellular infiltration, and airway responsiveness in a murine model of allergic asthma. Am J Respir Cell Mol Biol. 1998;19(5):826–35. doi: 10.1165/ajrcmb.19.5.3027 9806748.

41. Girdlestone J, Wing M. Autocrine activation by interferon-gamma of STAT factors following T cell activation. Eur J Immunol. 1996;26(3):704–9. doi: 10.1002/eji.1830260329 8605941.

42. Normark J, Nelson M, Engstrom P, Andersson M, Bjork R, Moritz T, et al. Maladjusted host immune responses induce experimental cerebral malaria-like pathology in a murine Borrelia and Plasmodium co-infection model. PLoS One. 2014;9(7):e103295. doi: 10.1371/journal.pone.0103295 25075973; PubMed Central PMCID: PMC4116174.

43. Nacher M, Singhasivanon P, Traore B, Vannaphan S, Gay F, Chindanond D, et al. Helminth infections are associated with protection from cerebral malaria and increased nitrogen derivatives concentrations in Thailand. Am J Trop Med Hyg. 2002;66(3):304–9. doi: 10.4269/ajtmh.2002.66.304 12139225.

44. Egima CM, Macedo SF, Sasso GR, Covarrubias C, Cortez M, Maeda FY, et al. Co-infection with Trypanosoma cruzi protects mice against early death by neurological or pulmonary disorders induced by Plasmodium berghei ANKA. Malar J. 2007;6:90. doi: 10.1186/1475-2875-6-90 17620126; PubMed Central PMCID: PMC1965473.

45. Stijlemans B, Caljon G, Van Den Abbeele J, Van Ginderachter JA, Magez S, De Trez C. Immune Evasion Strategies of Trypanosoma brucei within the Mammalian Host: Progression to Pathogenicity. Front Immunol. 2016;7:233. doi: 10.3389/fimmu.2016.00233 27446070; PubMed Central PMCID: PMC4919330.

46. Amole BO, Clarkson AB Jr., Shear HL. Pathogenesis of anemia in Trypanosoma brucei-infected mice. Infect Immun. 1982;36(3):1060–8. 7201455; PubMed Central PMCID: PMC551439.

47. Anosa VO, Kaneko JJ. Pathogenesis of Trypanosoma brucei infection in deer mice (Peromyscus maniculatus). Ultrastructural pathology of the spleen, liver, heart, and kidney. Vet Pathol. 1984;21(2):229–37. doi: 10.1177/030098588402100216 6730206.

48. Brun R, Blum J, Chappuis F, Burri C. Human African trypanosomiasis. Lancet. 2010;375(9709):148–59. doi: 10.1016/S0140-6736(09)60829-1 19833383.

49. Stijlemans B, De Baetselier P, Magez S, Van Ginderachter JA, De Trez C. African Trypanosomiasis-Associated Anemia: The Contribution of the Interplay between Parasites and the Mononuclear Phagocyte System. Front Immunol. 2018;9:218. doi: 10.3389/fimmu.2018.00218 29497418; PubMed Central PMCID: PMC5818406.

50. Rijo-Ferreira F, Carvalho T, Afonso C, Sanches-Vaz M, Costa RM, Figueiredo LM, et al. Sleeping sickness is a circadian disorder. Nat Commun. 2018;9(1):62. doi: 10.1038/s41467-017-02484-2 29302035; PubMed Central PMCID: PMC5754353.

51. De Muylder G, Daulouede S, Lecordier L, Uzureau P, Morias Y, Van Den Abbeele J, et al. A Trypanosoma brucei kinesin heavy chain promotes parasite growth by triggering host arginase activity. PLoS Pathog. 2013;9(10):e1003731. doi: 10.1371/journal.ppat.1003731 24204274; PubMed Central PMCID: PMC3814429.

52. Dempsey WL, Mansfield JM. Lymphocyte function in experimental African trypanosomiasis. V. Role of antibody and the mononuclear phagocyte system in variant-specific immunity. J Immunol. 1983;130(1):405–11. 6847889.

53. Albright JW, Long GW, Albright JF. The liver as a major site of immunological elimination of murine trypanosome infection, demonstrated with the liver perfusion model. Infect Immun. 1990;58(6):1965–70. 2341187; PubMed Central PMCID: PMC258751.

54. Bosschaerts T, Guilliams M, Stijlemans B, De Baetselier P, Beschin A. Understanding the role of monocytic cells in liver inflammation using parasite infection as a model. Immunobiology. 2009;214(9–10):737–47. doi: 10.1016/j.imbio.2009.06.010 19577324.

55. Guicciardi ME, Malhi H, Mott JL, Gores GJ. Apoptosis and necrosis in the liver. Compr Physiol. 2013;3(2):977–1010. doi: 10.1002/cphy.c120020 23720337; PubMed Central PMCID: PMC3867948.

56. Caljon G, Mabille D, Stijlemans B, De Trez C, Mazzone M, Tacchini-Cottier F, et al. Neutrophils enhance early Trypanosoma brucei infection onset. Sci Rep. 2018;8(1):11203. doi: 10.1038/s41598-018-29527-y 30046157; PubMed Central PMCID: PMC6060092.

57. Miller JL, Sack BK, Baldwin M, Vaughan AM, Kappe SHI. Interferon-mediated innate immune responses against malaria parasite liver stages. Cell Rep. 2014;7(2):436–47. doi: 10.1016/j.celrep.2014.03.018 24703850.

58. Moriyasu T, Nakamura R, Deloer S, Senba M, Kubo M, Inoue M, et al. Schistosoma mansoni infection suppresses the growth of Plasmodium yoelii parasites in the liver and reduces gametocyte infectivity to mosquitoes. PLoS Negl Trop Dis. 2018;12(1):e0006197. doi: 10.1371/journal.pntd.0006197 29373600; PubMed Central PMCID: PMC5802944.

59. Lopez R, Demick KP, Mansfield JM, Paulnock DM. Type I IFNs play a role in early resistance, but subsequent susceptibility, to the African trypanosomes. J Immunol. 2008;181(7):4908–17. doi: 10.4049/jimmunol.181.7.4908 18802094; PubMed Central PMCID: PMC2582636.

60. Murray PK, Jennings FW, Murray M, Urquhart GM. The nature of immunosuppression in Trypanosoma brucei infections in mice. I. The role of the macrophage. Immunology. 1974;27(5):815–24. 4435838; PubMed Central PMCID: PMC1445664.

61. Shi M, Pan W, Tabel H. Experimental African trypanosomiasis: IFN-gamma mediates early mortality. Eur J Immunol. 2003;33(1):108–18. doi: 10.1002/immu.200390013 12594839.

62. Shi M, Wei G, Pan W, Tabel H. Trypanosoma congolense infections: antibody-mediated phagocytosis by Kupffer cells. J Leukoc Biol. 2004;76(2):399–405. doi: 10.1189/jlb.1003500 15136584.

63. Boonhok R, Rachaphaew N, Duangmanee A, Chobson P, Pattaradilokrat S, Utaisincharoen P, et al. LAP-like process as an immune mechanism downstream of IFN-gamma in control of the human malaria Plasmodium vivax liver stage. Proc Natl Acad Sci U S A. 2016;113(25):E3519–28. doi: 10.1073/pnas.1525606113 27185909; PubMed Central PMCID: PMC4922146.

64. Yoshida A, Maruyama H, Kumagai T, Amano T, Kobayashi F, Zhang M, et al. Schistosoma mansoni infection cancels the susceptibility to Plasmodium chabaudi through induction of type 1 immune responses in A/J mice. Int Immunol. 2000;12(8):1117–25. doi: 10.1093/intimm/12.8.1117 10917886.

65. Salazar-Castanon VH, Juarez-Avelar I, Legorreta-Herrera M, Govezensky T, Rodriguez-Sosa M. Co-infection: the outcome of Plasmodium infection differs according to the time of pre-existing helminth infection. Parasitol Res. 2018;117(9):2767–84. doi: 10.1007/s00436-018-5965-9 29938323.

66. King T, Lamb T. Interferon-gamma: The Jekyll and Hyde of Malaria. PLoS Pathog. 2015;11(10):e1005118. doi: 10.1371/journal.ppat.1005118 26426121; PubMed Central PMCID: PMC4591348.

67. Mitchell AJ, Hansen AM, Hee L, Ball HJ, Potter SM, Walker JC, et al. Early cytokine production is associated with protection from murine cerebral malaria. Infect Immun. 2005;73(9):5645–53. doi: 10.1128/IAI.73.9.5645-5653.2005 16113282; PubMed Central PMCID: PMC1231146.

68. Villegas-Mendez A, Greig R, Shaw TN, de Souza JB, Gwyer Findlay E, Stumhofer JS, et al. IFN-gamma-producing CD4+ T cells promote experimental cerebral malaria by modulating CD8+ T cell accumulation within the brain. J Immunol. 2012;189(2):968–79. doi: 10.4049/jimmunol.1200688 22723523; PubMed Central PMCID: PMC3393641.

69. Cardillo F, Voltarelli JC, Reed SG, Silva JS. Regulation of Trypanosoma cruzi infection in mice by gamma interferon and interleukin 10: role of NK cells. Infect Immun. 1996;64(1):128–34. 8557330; PubMed Central PMCID: PMC173737.

70. Doolan DL, Hoffman SL. The complexity of protective immunity against liver-stage malaria. J Immunol. 2000;165(3):1453–62. doi: 10.4049/jimmunol.165.3.1453 10903750.

71. Mendes AM, Scholzen A, Mueller A-K, Shahid M. Khan, Sauerwein RW, Prudêncio M. Whole-Sporozoite Malaria Vaccines. In: Mota MM, Rodriguez A, editors. Malaria. Cham, Switzerland: Springer; 2017.

72. Beeson JG, Kurtovic L, Dobano C, Opi DH, Chan JA, Feng G, et al. Challenges and strategies for developing efficacious and long-lasting malaria vaccines. Sci Transl Med. 2019;11(474). doi: 10.1126/scitranslmed.aau1458 30626712.

73. Engstler M, Boshart M. Cold shock and regulation of surface protein trafficking convey sensitization to inducers of stage differentiation in Trypanosoma brucei. Genes Dev. 2004;18(22):2798–811. doi: 10.1101/gad.323404 15545633; PubMed Central PMCID: PMC528899.

74. Franke-Fayard B, Trueman H, Ramesar J, Mendoza J, van der Keur M, van der Linden R, et al. A Plasmodium berghei reference line that constitutively expresses GFP at a high level throughout the complete life cycle. Mol Biochem Parasitol. 2004;137(1):23–33. doi: 10.1016/j.molbiopara.2004.04.007 15279948.

75. Ploemen IH, Prudencio M, Douradinha BG, Ramesar J, Fonager J, van Gemert GJ, et al. Visualisation and quantitative analysis of the rodent malaria liver stage by real time imaging. PLoS One. 2009;4(11):e7881. doi: 10.1371/journal.pone.0007881 19924309; PubMed Central PMCID: PMC2775639.

76. Lin JW, Annoura T, Sajid M, Chevalley-Maurel S, Ramesar J, Klop O, et al. A novel 'gene insertion/marker out' (GIMO) method for transgene expression and gene complementation in rodent malaria parasites. PLoS One. 2011;6(12):e29289. doi: 10.1371/journal.pone.0029289 22216235; PubMed Central PMCID: PMC3246482.

77. Carroll RW, Wainwright MS, Kim KY, Kidambi T, Gomez ND, Taylor T, et al. A rapid murine coma and behavior scale for quantitative assessment of murine cerebral malaria. PLoS One. 2010;5(10). doi: 10.1371/journal.pone.0013124 20957049; PubMed Central PMCID: PMC2948515.

78. Liehl P, Zuzarte-Luis V, Chan J, Zillinger T, Baptista F, Carapau D, et al. Host-cell sensors for Plasmodium activate innate immunity against liver-stage infection. Nat Med. 2014;20(1):47–53. doi: 10.1038/nm.3424 24362933; PubMed Central PMCID: PMC4096771.

79. Mendes AM, Albuquerque IS, Machado M, Pissarra J, Meireles P, Prudencio M. Inhibition of Plasmodium Liver Infection by Ivermectin. Antimicrob Agents Chemother. 2017;61(2). doi: 10.1128/AAC.02005-16 27895022; PubMed Central PMCID: PMC5278742.

80. Bruna-Romero O, Hafalla JC, Gonzalez-Aseguinolaza G, Sano G, Tsuji M, Zavala F. Detection of malaria liver-stages in mice infected through the bite of a single Anopheles mosquito using a highly sensitive real-time PCR. Int J Parasitol. 2001;31(13):1499–502. doi: 10.1016/s0020-7519(01)00265-x 11595237.

81. Meireles P, Sales-Dias J, Andrade CM, Mello-Vieira J, Mancio-Silva L, Simas JP, et al. GLUT1-mediated glucose uptake plays a crucial role during Plasmodium hepatic infection. Cell Microbiol. 2017;19(2). doi: 10.1111/cmi.12646 27404888; PubMed Central PMCID: PMC5297879.