Comparative studies of serum-free media and detection techniques for in vitro drug sensitivity assessment of Plasmodium falciparum

Abstract

Malaria continues to be a devastating disease. In a previous study, we formulated a chemically defined culture medium that is able to sustain the complete intraerythrocytic growth of Plasmodium falciparum. We tested the feasibility of using the medium (CDRPMI) as well as human serum-free media enriched with commercially available human-serum substitutes (GFSRPMI and ALBRPMI) to assess the drug sensitivity of P. falciparum, using chloroquine diphosphate (CQ) and dihydroartemisinin (DHART) as conventional antimalarial drugs. Growth inhibition was measured by four different methods: flow cytometry with SYBR Green I (FCM), microscopy (Giemsa method), enzymatic estimation of parasite lactate dehydrogenase (pLDH), and histidine-rich protein 2 (HRPII) determination. In drug sensitivity tests on asynchronous parasites cultured for 96 h in the presence of drugs, the dose-response curves were similar and differences in the 50% growth inhibition concentrations for the drugs, which were estimated by the four methods, were not statistically significant for the three culture media. The effect of the drugs on the growth of synchronous parasites at the ring stage was also assessed in micro-volume tests by three different methods of FCM: tracking fluorescent erythrocytes, schizont test, and merozoite test. Dose-response curves for the drugs were similar, and differences in the 50% growth inhibition concentrations were not statistically significant for CDRPMI and GFSRPMI. Thus CDRPMI as well as GFSRPMI and ALBRPMI can be similarly useful media for drug sensitivity testing of P. falciparum. The FCM, pLDH and HRPII estimations were fast and reliable detection methods, with FCM allowing schizont and merozoite tests to be performed with shorter periods of culture.

Share and Cite:

Kwansa-Bentum, B. , Izumiyama, S. , Kitamura, K. , Obata-Ninomiya, K. , Ohta, N. and Asahi, H. (2013) Comparative studies of serum-free media and detection techniques for in vitro drug sensitivity assessment of Plasmodium falciparum. Open Journal of Clinical Diagnostics, 3, 115-121. doi: 10.4236/ojcd.2013.33020.

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1] World Health Organization (2012) World malaria report 2012. WHO Press, Geneva. http://www.who.int/malaria/publications/world_malaria_report_2012/wmr2012_factseet.pdf
[2] Ridley, R.G. (2002) Medical need, scientific opportunity and the drive for antimalarial drugs. Nature, 415, 686-693. doi:10.1038/415686a
[3] Trager, W. and Jensen, J.B. (1997) Continuous culture of Plasmodium falciparum: Its impact on malaria research. International Journal for Parasitology, 27, 989-1006. doi:10.1016/S0020-7519(97)00080-5
[4] Asahi, H. and Kanazawa, T. (1994) Continuous cultivation of intraerythrocytic Plasmodium falciparum in a serum-free medium with the use of a growth-promoting factor. Parasitology, 109, 397-401. doi:10.1017/S0031182000080641
[5] Cranmer, S.L., Magowan, C., Liang, J., Coppel, R.L. and Cooke, B.M. (1997) An alternative to serum for cultivation of Plasmodium falciparum in vitro. Transactions of the Royal Society of Tropical Medicine and Hygiene, 91, 363-365. doi:10.1016/S0035-9203(97)90110-3
[6] Kim, Y.J., et al. (2012) Development of a chemically defined minimal medium for the exponential growth of Leuconostocmesenteroides ATCC8293. Journal of Microbiology and Biotechnology, 22, 1518-1522. doi:10.4014/jmb.1205.05053
[7] Mimura, S., et al. (2011) Growth factor-defined culture medium for human mesenchymal stem cells. International Journal of Developmental Biology, 55, 181-187. doi:10.1387/ijdb.103232sm
[8] Asahi, H. (2009) Plasmodium falciparum: Chemically defined medium for continuous intraerythrocytic growth using lipids and recombinant albumin. Experimental Parasitology, 121, 22-28. doi:10.1016/j.exppara.2008.09.009
[9] Makler, M.T. and Hinrichs, D.J. (1993) Measurement of the lactate dehydrogenase activity of Plasmodium falciparum as an assessment of parasitemia. The American Jornal of Tropical Medicine and Hygiene, 48, 205-210.
[10] Druilhe, P., Moreno, A., Blanc, C., Brasseur, P.H. and Jacquier, P. (2001) A colorimetric in vitro drug sensitivity assay for Plasmodium falciparum based on a highly sensitive double-site lactate dehydrogenase antigen-capture enzyme-linked immunosorbent assay. The American Journal of Tropical Medicine and Hygiene, 64, 233-241.
[11] Noedl, H., Wongsrichanalai, C. and Wernsdorfer, W.H. (2003) Malaria drug-sensitivity testing: New assays, new perspectives. Trends in Parasitology, 19, 175-181. doi:10.1016/S1471-4922(03)00028-X
[12] Asahi, H., Kanazawa, T., Hirayama, N. and Kajihara, Y. (2005) Investigating serum factors promoting erythrocytic growth of Plasmodium falciparum. Experimental Parasitology, 109, 7-15. doi:10.1016/j.exppara.2004.10.002
[13] Piper, R.C., Buchanan, I., Choi, Y.H. and Makler, M.T. (2011) Opportunities for improving pLDH-based malaria diagnostic tests. Malaria journal, 10, 213. doi:10.1186/1475-2875-10-213
[14] Izumiyama, S., Omura, M., Takasaki, T., Ohmae, H. and Asahi, H. (2009) Plasmodium falciparum: Development and validation of a measure of intraerythrocytic growth using SYBR Green I in a flow cytometer. Experimental Parasitology, 121, 144-150. doi:10.1016/j.exppara.2008.10.008
[15] Bei, A.K., et al. (2010) A flow cytometry-based assay for measuring invasion of red blood cells by Plasmodium falciparum. American Journal of Hematology, 85, 234-237. doi:10.1002/ajh.21642
[16] Somsak, V., Srichairatanakool, S., Yuthavong, Y., Kamchonwongpaisan, S. and Uthaipibull, C. (2012) Flow cytometric enumeration of Plasmodium berghei-infected red blood cells stained with SYBR Green I. Acta tropica, 122, 113-118. doi:10.1016/j.actatropica.2011.12.010
[17] Asahi, H., Izumiyama, S., Tolba, M.E. and Kwansa-Bentum, B. (2011) Plasmodium falciparum: Differing effects of non-esterified fatty acids and phospholipids on intraerythrocytic growth in serum-free medium. Experimental Parasitology, 127, 708-713. doi:10.1016/j.exppara.2010.11.001
[18] Asahi, H. (2012) Intraerythrocytic Plasmodium falciparum Growth in Serum-Free Medium with an Emphasis on Growth-Promoting Factors. In: Okwa, O.O., Ed., Malaria Parasites, InTech, Croatia, 73-90. doi:10.5772/33212
[19] Asahi, H., Tolba, M.E.M., Tanabe, M. and Ohmae, H. (2013) Molecular factors that are associated with early developmental arrest of intraerythrocytic Plasmodium falciparum. Canadian Journal of Microbiology, 59, 485-493. doi:10.1139/cjm-2013-0166
[20] Grimberg, B.T. (2011) Methodology and application of flow cytometry for investigation of human malaria parasites. Journal of Immunological Methods, 367, 1-16. doi:10.1016/j.jim.2011.01.015
[21] Bouillon, A., Gorgette, O., Mercereau-Puijalon, O. and Barale, J.C. (2013) Screening and evaluation of inhibitors of Plasmodium falciparum merozoite egress and invasion using cytometry. In: Menard, R., Ed., Malaria: Methods and Protocols, Methods in Molecular Biology, Springer Science + Business Media, 523-534.
[22] Dondorp, A.M., et al. (2009) Artemisinin resistance in Plasmodium falciparum malaria. The New England Journal of Medicine, 361, 455-467. doi:10.1056/NEJMoa0808859
[23] World Health Organization (2011) Global plan for artemisinin resistance containment. WHO press, Geneva.
[24] Kwansa-Bentum, B., et al. (2011) Administrative practices of health professionals and use of artesunate-amodiaquine by community members for treating uncomplicated malaria in southern Ghana: Implications for artemisinin-based combination therapy deployment. Tropical Medicine and International Health, 16, 1215-1224. doi:10.1111/j.1365-3156.2011.02833.x
[25] Kwansa-Bentum, B., et al. (2011) Plasmodium falciparum isolates from southern Ghana exhibit polymorphisms in the SERCA-type PfATPase6 though sensitive to artesunate in vitro. Malaria Journal, 10, 187. http://www.malariajournal.com/content/10/1/187 doi:10.1186/1475-2875-10-187

Copyright © 2024 by authors and Scientific Research Publishing Inc.

Creative Commons License

This work and the related PDF file are licensed under a Creative Commons Attribution 4.0 International License.