Susceptibility of Thai isolates of Plasmodium falciparum to artemisinine (qinghaosu) and artemether

Bulletin ofthe World Health Organization, 63 (3): 617-619 (1985) © World Health Organization 1985 Susceptibility of Thai isolates of Plasmodium falciarum to artemisinine (qinghaosu) and artemether SODSRI THAITHONG1 & G. H. BEALE2 Eleven Thai isolates and one West African isolate of Plasmodium falciparum were tested for their susceptibility to the Chinese antimalarial drugs artemisinine (qinghaosu) and artemether. The isolates were cultivated by the Trager-Jensen candle-jar technique and exposed to the action of the drugsfor 36-48 hours. Artemisinine inhibited growth ofmost isolates at 10 -7_i08 mol/litre and artemether at 10- 8 mol/litre (with an initial para- sitaemia of O.5-1.O%0o). Slight variation in the sensitivity ofdifferent isolates wasfound, but there was no correlation between sensitivity to artemisinine or artemether and sensitivity to pyrimethamine, pyrimethamine/sulfadoxine, or chloroquine. The action of artemisinine and artemether was reduced when the initial parasitaemia of the treated cultures was raised. Since most Thai isolates of Plasmodium fal- ciparum are now highly resistant to pyrimethamine, pyrimethamine/sulfadoxine, and chloroquine and seem to be developing resistance to quinine, it was thought desirable to determine the susceptibility of various Thai isolates to the Chinese antimalarial drugs, artemisinine (qinghaosu) and artemether.a MATERIALS AND METHODS The isolates tested consisted of two uncloned isolates from Kanchanaburi (K1 and K31) and nine clones of an isolate from Mae Sod, Thailand (T9). In addition, one uncloned African isolate (GI) from the Gambia was tested for comparison. The parasites were tested for susceptibility to artemisinine and artemether by an in vitro method similar to that described previously (1, 2) for use with other antimalarial drugs. Prior to testing, the isolates were maintained in culture in Petri dishes, by the candle-jar method (3), in complete RPMI 1640 medium with 100 ml/litre serum. Samples containing a range of drug concentrations, from 10- 7 mol/litre to 10- 12 mol/litre, were placed in the wells of micro- titration plates and incubated at 37 °C for 36-72 h. Changes of medium (containing drugs) were made Department of Biology, Faculty of Science, Chulalongkorn University, Bangkok, Thailand. 2 Institute of Animal Genetics, Edinburgh, EH 9, Scotland. a Unpublished WHO document, TDR/CHEMAL-SWG(4)/ QHS/81.3, 1981. after 24 h and after 48 h. At the end of the drug exposure periods, Giemsa-stained thin smears were made and examined under the microscope for the presence of parasites. The results are expressed in terms of the minimum inhibitory concentration (MIC), i.e., that concentration of drug, expressed as molar units, capable of completely suppressing growth of the parasites. The drugs were kindly supplied by the Chinese authorities and made available to the authors through the World Health Organization, Geneva. The drugs were dissolved in N, N-dimethyl formamide at a con- centration of 10 mol/litre. Tests showed that the solvent had no effect on parasite growth, at the highest concentration used (100 il in 10 ml of medium). RESULTS AND DISCUSSION Effect of initial parasitaemia Preliminary tests showed that the MIC values obtained depended on the initial parasitaemia of the cultures in the wells, as shown in Table 1. Hence in the main tests, the initial parasitaemia was kept within the range 0.5-1 %lo. Effect of exposure time It was found that exposure to the drugs for 36, 48, or 72 hours did not affect the MIC values. In the main 4561 617- S. THAITHONG & G. H. BEALE Table 1. Effect of initial parasitaemia on parasite survival after treatment with artemisinine (qinghaosu) and artemether (isolate K,) MIC value (mol/litre) Initial Artemisinine Artemether parasitaemia (%) (qinghaosu) 2 10- io0-7 1 10-710- 10-8 0.5 108 10o8 0.25 10 10-9 0.1 10 - 10 -9 tests, therefore, the exposure time was standardized at 36 hours. Sensitivity of isolates Table 2 shows the MIC values for artemisinine and artemether for eleven Thai isolates and one West African isolate (G,). The sensitivity of the same isolates to three other drugs-pyrimethamine, pyrimethamine/sulfadoxine, and chloroquine -is also shown. Most of the isolates tested showed approximately the same sensitivity to artemisinine and artemether, though some minor variations were observed. Both drugs were equally effective against isolates that varied considerably in their sensitivity to pyri- methamine, pyrimethamine/sulfadoxine, and chloro- quine. CONCLUSIONS From the tests reported here, it may be concluded that artemisinine (qinghaosu) and artemether were highly effective against all the isolates of P.fal- ciparum tested, irrespective of their sensitivity or resistance to pyrimethamine, pyrimethamine/sul- fadoxine, or chloroquine. There was minor variation in the susceptibility of some isolates to both artemi- sinine and artemether. Exposure to artemisinine or artemether for only 36 hours produced as great an effect as for 48 or 72 hours. Since the cultures used in these tests contained unsynchronized parasites, this is interpreted to imply that all asexual stages of the erythrocytic parasites are equally sensitive. Artemisinine (qinghaosu) was found to be slightly less effective than artemether, in terms of MIC, but the difference was quite minor. The inhibitory effect of the two drugs was reduced when the initial parasitaemia was high. Table 2. MIC values of artemisinine (qinghaosu) and artemether for 11 Thai and 1 African isolate of P. falciparum Isolate" Place of origin MIC (mol/litre)a Susceptibility to:b Artemisinine Artemether Pyrimethamine Pyrimethamine/ Chloroquine sulfadoxine K, Kanchanaburi 10-,o0-,8 10-8 R R R K31 Kanchanaburi l0o-,-lo-0 lo8 5 5 S Tg997 Mae Sod 10-,lo-8 10-7_10-8 R R R Tg-98 Mae Sod 10-7_10-8 10-8 R R S Tg9_99 Mae Sod 10-7_10-1 10-8 R R R* Tg-loo Mae Sod 10-7_10-8 108 R R R Tg 102 Mae Sod 10-8 10-7_10-8 R R R Tg-103 Mae Sod 10-7_10-8 108 NT S NT T9_jo5 Mae Sod 10-7_10-8 108 R R R Tq-m,0Mae Sod 10-7-10-8 1o-8 R R R Tg-107 Mae Sod 10-7_10-8 10-7_10-8 S S S G, Gambia l0o' 10-8 S S S a T9.97-T91e07 are clones derived from isolate Tg. bR = resistant; S = sensitive; R * = intermediate; NT = not tested (3, 4). 618 SUSCEPTIBILITY OF P. FALCIPARUM TO ARTEMISININE AND ARTEMETHER 619 ACKNOWLEDGEMENTS We wish to express our thanks to the Chinese authorities for supplying the samples of artemisinine and artemether and to Dr P. 1. Trigg, World Health Organization, Geneva, for making the necessary arrangements. This work was supported by grants from the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases, the Institute of Health Research, Chulalongkorn University, and the Wellcome Trust. RESUME SENSIBILITE D'ISOLEMENTS THAILANDAIS DE PLASMODIUM FALCIPARUM VIS-A-VIS DE L'ARTEMISININE (QINGHAOSU) ET DE L'ARTEMETHER Le pr6sent article decrit les reactions in vitro de deux isole- ments thalilandais non clones de Plasmodiumfalciparum, de neuf clones d'un troisieme isolement thailandais et d'un isolement africain, vis-a-vis de deux antipalud6ens chinois, l'artemisinine (qinghaosu) et l'artemether. Les parasites ont e mis en contact avec ces m6dicaments pendant 36 heures a 37 IC, dans du RPMI 1640 additionne de 100 ml/l de serum; les etalements minces colores au Giemsa ont ensuite 6t6 examines au microscope. Les resultats sont exprimes d'apres la concentration minimale inhibitrice (CMI), c'est- a-dire la concentration minimale qui inhibe totalement le developpement du parasite. La parasit6mie initiale des cultures a et maintenue entre 0,5 et 1%. On a constate que la CMI pour les deux medicaments se situait dans l'intervalle I0-8_10-7 mol/litre. Seules de legeres variations de la CMI ont e observees entre les divers isole- ments alors, pourtant, que les isolements etudies differaient fortement par leur sensibilite vis-a-vis d'autres substances comme la pyrimethamine, la pyrim6thamine/sulfadoxine et la chloroquine. L'effet de l'artemisinine et de l'art6mether diminuait lorsque la parasitemie initiale des cultures traitees 6tait augment&e. On obtenait les memes resultats en expo- sant le parasite a l'art6m6ther pendant 36, 48 ou 72 heures. Etant donne que les cultures employ6es dans ces essais etaient constitu6es de parasites a des stades differents, on en conclut que tous les stades asexues du cycle erythrocytaire sont egalement sensibles a ces medicaments. REFERENCES 1. THAITHONG, S. & BEALE, G. H. Transactions of the Royal Society of Tropical Medicine and Hygiene, 75: 271-273 (1981). 2. THAITHONG, S. ET AL. Transactions of the Royal Society of Tropical Medicine and Hygiene, 77: 228-231 (1983). 3. TRAGER, W. & JENSEN, J. B. Science, 193: 674-675 (1976). 4. THAITHONG, S. ET AL. Transactions of the Royal Society of Tropical Medicine and Hygiene, 78: 242-245 (1984).