Open Access
Research (Published online: 19-07-2017)
14. Phenotypic approach artemisinin resistance in malaria rodent as in vivo model
Lilik Maslachah, Thomas V. Widiyatno, Lita Rakhma Yustinasari and Hani Plumeriastuti
Veterinary World, 10(7): 790-797

Lilik Maslachah: Department of Basic Medicine, Veterinary Pharmacy Laboratory, Faculty of Veterinary Medicine, Airlangga University Surabaya, Indonesia.
Thomas V. Widiyatno: Department of Pathology, Faculty of Veterinary Medicine, Airlangga University Surabaya, Indonesia.
Lita Rakhma Yustinasari: Department of Veterinary Anatomy, Faculty of Veterinary Medicine, Airlangga University Surabaya, Indonesia.
Hani Plumeriastuti: Department of Veterinary Pathology, Faculty of Veterinary Medicine, Airlangga University Surabaya, Indonesia.

doi: 10.14202/vetworld.2017.790-797

Share this article on [Facebook] [LinkedIn]

Article history: Received: 14-01-2017, Accepted: 05-06-2017, Published online: 19-07-2017

Corresponding author: Lilik Maslachah


Citation: Maslachah L, Widiyatno TV, Yustinasari LR, Plumeriastuti H (2017) Phenotypic approach artemisinin resistance in malaria rodent as in vivo model, Veterinary World, 10(7): 790-797.

Aim: The aim of this study is to prove the development of artemisinin resistance phenotypically in malaria rodent as an in vivo resistance development model in humans.

Materials and Methods: Plasmodium berghei was infected intraperitoneally in mice, then artemisinin was given with "4-day-test" with effective dose (ED) 99% dose for 3 days which begins 48 h after infection (D2, D3, and D4). Parasite development was followed during 5th until 10th days of infection. After parasitemia >2% of red blood cell which contains parasites on 1 mice, that mice were used as donor to be passaged on the new 5 mice. After that, parasitemia was calculated. ED50 and ED90 were examined with parasite clearance time (PCT), recrudescence time (RT), and also morphology development examination of intraerythrocytic cycle of P. berghei with transmission electron microscope.

Results: Among the control group compare with the treatment group showed significant differences at α=0.05 on 5th day (D5) until 10th day (D10). The control group of 4th passage (K4) with passage treatment group of 4th passage (P4) on the 10th days (D10) post infection showed no significant differences in the α=0.05. The average percentage of inhibition growth was decreasing which is started from 5th to 10th day post infection in P1, P2, P3, and P4. On the development of P. berghei stage, which is given repeated artemisinin and repeated passage, there was a formation of dormant and also vacuoles in Plasmodium that exposed to the drug.

Conclusion: Exposure to artemisinin with repeated passages in mice increased the value of ED50 and ED90, decreased the PCT and RT and also changes in morphology dormant and vacuole formation.

Keywords: artemisinin, parasite clearance time, phenotypic, Plasmodium berghei, recrudescence time, resistance.


1. Afonso, A., Hunt, P., Cheesman, S., Alves, A.C., Cunha, C.V., do Rosario, V. and Cravo, P. (2006) Malaria parasites can develop stable resistance to artemisinin but lack mutations in candidate genes atp6 (encoding the sarcoplasmic and endoplasmic reticulum Ca2+ATPase) tctp, mdr1 and cg10. Antimicrob. Agents Chemother., 61(7): 480-489. [Crossref] [PubMed] [PMC]

2. Noedl, H. (2008) Evidence of artemisinin-resistant malaria in Western Cambodia. N. Engl. J. Med., 359(24): 2619-2620. [Crossref] [PubMed]

3. Wongsrichanalai, C. and Meshnick, S.R. (2008) Declining artesunat-mefloquine efficacy against falciparum malaria on Cambodia-Thailand border. Emerg. Infect. Dis., 4(5): 716-718. [Crossref] [PubMed] [PMC]

4. Maslachah, L. (2013) Effect of Repeated Exposure of Artemisinin towards Plasmodium falcifarum Resistance Development in vitro. Dissertation Airlangga University.

5. Craig, A.G., Grau, C.G., Janse, C., James, W., Kazura, J.W., Milner, D., Barnwell, J.W., Turner,G. and Langhorne, J. (2012) The role of animal models for research on severe malaria. PLoS Pathog., 8(2): e1002401. [Crossref]

6. Muregi, F.W., Ohta, I., Masato, U., Kino, H. and Ishih, A. (2011) Resistance of a rodent malaria parasite to a thymidylate synthase inhibitor induces an apoptotic parasite death and imposes a huge coat of fitness. Plos One, 6(6): e21251. [Crossref]

7. Kiboi, D.M., Irungu, B.N., Langat, B., Wittlin, S., Brun, R., Chollet, J., Abiodun, O. and Nganga, J.K. (2009) Plasmodium berghei ANKA: Selection of resistance to piperaquine and lumefantrine in a mouse model. Exp. Parasitol., 122: 196-202. [Crossref]

8. Henriques, G., Martinelli, A., Rodrigues, L., Modrzynka, K., Fawcett, R. and Houston, D.R. (2013) Artemisinin resistance inrodentmalaria - Mutation in the AP2 adaptor M-chain suggest involement of endocytosis and membrane protein trafficking. Malar. J., 12: 118. [Crossref] [PubMed] [PMC]

9. Garcia, C.R., de Azevedo, M.F., Wunderlich, G., Budu, A., Young, J.A. and Bannister, L. (2007) Plasmodium in the post genomic era: New insight into molecular cell biology of malaria parasites. Int. Rev. Cell Mol. Biol., 266: 85-156. [Crossref]

10. Moll, K., Kaneko, K., Scherf, A. and Wahlgren, M. (2013) Methods in Malaria Research. 6th ed. MR4/ATCC, UK. p1-464.

11. Kimani, S.K., Ng'ang'a, J.K., Kariuki, D.W., Kinjua, J. and Kiboi, D.M. (2014) Plasmodium berghe ANKA: Selection of pyronaridine resistance in mouse model. Afr. J. Biochem. Res., 12: 111-117. [Crossref]

12. La Crue, A.N., Scheel, M., Kennedy, K., Kumar, N. and Kyle, D.E. (2011) Effects of artesunate on parasite recrudescence and dormancy in rodent malaria model Plasmodium vinckei. Plos One, 6(10): e26689. [Crossref] [PubMed] [PMC]

13. Sanz, L.M., Crespo, B., De-Cozar, C., Ding, X.C., Liergo, J.L., Burrows, J.N., Garcia-Butos, J.F. and Gamo, F.J. (2012) P. falciparum in vitro killing rates allow to discriminate between different antimalarial mode of action. Plos One, 7(2): e30949. [Crossref] [PubMed] [PMC]

14. Veiga, M.I., Ferreira, P.E., Schmidt, B.A., Schmidt, B.A., Ribacke, U., Bjorkman, A., Tichopad, A. and Gil, J.P. (2010) Antimalarial exposure delays Plasmodium falciparum intraerytrocytic cycle and drives drug transporter genes expression. Plos One, 5(8): e12408. [Crossref] [PubMed] [PMC]

15. Crespo, M.P., Avery, T.D., Hanssen, E., Fox, E., Robinson, T.V., Valente, P. and Tilley, L.T. (2008) Artemisinin Anda series of novel endoperoxide antimalarials exert early effects on digestive vacuole morphology. Antimicrob. Agents Chemother., 52(1): 98-109. [Crossref] [PubMed] [PMC]

16. Beez, D., Sanchez, C.P., Stein,W.D. and Lanzer, M. (2010) Genetic predisposition favors the acquisition of stable artemisinin resistance in malaria parasites. Antimicrob. Agents Chemother., 55(1): 50-55. [Crossref] [PubMed] [PMC]

17. Cheng, Q., Kyle, D.E. and Gatton, M.L. (2012) Artemisinin resistance in Plasmodium falciparum: A process linked to dormancy. Int. J. Parasitol., 2: 249-255. [Crossref]

18. Maslachah, L. (2015) Phenotypic profile of Plasmodium falciparum papua 2300 strain in vitro exposured with antimalarial artemisinin. MKB, 47(3): 129-136.

19. Witkowski, B., Lelievre, J., Barragan, M.J.L., Laurent, V., Su, X., Berry, A. and Vical, F.B. (2010) Increased tolerance to artemisinin in Plasmodium falciparum is mediated by a quiescence mechanism. Antimicrob. Agents Chemother., 54(5): 1872-1877. [Crossref] [PubMed] [PMC]

20. Tucker, M.S., Mutka, T., Sparks, K., Patel, J. and Kyle, D.E. (2012) Phenotypic and genotypic analysis of in vitro selected artemisinin resistent progeny of Plasmodium falciparum. Antimicrob. Agents Chemother., 56(1): 302-314. [Crossref] [PubMed] [PMC]

21. Chavchich, M., Gerena, L., Peters, J., Chen, N., Cheng, Q. and Kyle, D.E. (2010) Role of pfmdr1 amplification and expression in induction of resistance to artemisinin derivatives in Plasmodium falciparum. Antimicrob. Agents Chemother., 54: 2455-2464. [Crossref] [PubMed] [PMC]

22. Teuscher, F., Chen, N., Kyle, D.E., Gatton, M.L. and Cheng, Q. (2012) Phenotypic changes in artemisinin resistant Plasmodium falciparum line in vitro: Evidence for decreased sensitivity to dormancy and growth inhibition. Antimicrob. Agents Chemother., 56(1): 428-431. [Crossref] [PubMed] [PMC]

23. Teuscher, F., Gatton, M.L., Chen, N., Peters, J., Kyle, D.E. and Cheng, Q. (2010) Artemisinin induced dormancy in Plasmodium falciparum; duration, recovery rates, and implications intreatment failure. J. Infect. Dis., 202(9): 1362-1368. [Crossref] [PubMed] [PMC]