Plasmodium falciparum in continuous culture: a new medium for the in vitro test for sulfadoxine sensitivity*

Iulletin of the World Health Organization, 60 (3): 423 -426 (1982) Plasmodium falciparum in continuous culture: a new medium for the in vitro test for sulfadoxine sensitivity* CHARIYA R. BROCKELMAN' & PEERAPAN TAN-ARIYA2 The sulfadoxine sensitivity of two strains of Plasmodium falciparum from Thailand, FCM2 andFCMS, was assessed using two types ofculture medium, Waymouthformula and RPMI 1640. Growth of the parasite was completely inhibited by 0.5 mmol ofsulfadoxine per litre of Waymouth formula, whereas parasite growth in RPMI was not affected at this concentration. The apparent difference in drug sensitivity was shown to be caused by competition between 4-aminobenzoic acid and sulfadoxine. This hypothesis was further confirmed by the extent to which f4CJ-sulfadoxine was incorporated into the infected erythrocytes. The recent development of a method for continu- ous cultivation of Plasmodium falciparum in the laboratory (1) has permitted the study of the drug response of erythrocytic forms in vitro over a test period of more than one developmental cycle. The results obtained with chloroquine and pyrimethamine have shown good agreement with in vivo test methods (2, 3), but tests on sulfadoxine have required an un- realistically high concentration of the drug for mini- mal inhibition in vitro, although the parasite showed high in vivo susceptibility to sulfadoxine treatment. It has been suggested that this inconsistency is a result of competition between 4-aminobenzoic acid (PABA), a component of RPMI culture medium, and sulfa- doxine, a PABA analogue (3). It was thus necessary to find a medium that supports parasite growth but contains no PABA. Such a medium would permit further screening of sulfa drugs, which have become increasingly important in South-East Asia where chloroquine-resistant P.falciparum is now develop- ing resistance to pyrimethamine (3, 4). MATERIALS AND METHODS Parasites The P.falciparum lines used in this study, FCM2 and FCM5, were isolated from two residents of Trat, south-east Thailand, in September 1980, and have since been kept in continuous culture in type 0 human erythrocytes in a Petri dish and candle jar system, as * From the Department of Microbiology, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 4, Thailand. ' Associate Professor. 2 Research Associate. described by Trager & Jensen (1). Repeated experi- ments over a 3-month period showed that FCM2 was completely inhibited by 4 mmol and FCM5 by 12 mmol of sulfadoxine per litre of RPMI (3). Culture media The culture media used in this study were RPMI 1640 containing 1 mg of PABA per litre (5), and Waymouth powder formula which contained no PABA (5).a Each medium was constituted to contain 25 mmol of HEPES, 2 g of NaHCO3, and 100 ml of human serum per litre, the serum being collected from one individual. These media are designated complete RPMI (referred to as RP) and complete Waymouth (WM). In one series of experiments, 1 mg of PABA was added per litre of Waymouth, and used as a positive control. Determination ofPABA level Human serum and complete media containing serum were deproteinized (6) and aliquots of 2 ml were reacted with 4 ml of sodium nitrite solution (1 g/litre) and 0.4 ml of ammonium sulfamate solu- tion (5 g/litre). The mixture was coupled with 0.2 ml of an aqueous solution of N-naphthylethylene- diamine dihydrochloride (1 ml/litre).b The optical density of the resultant colour was measured in a Unicam spectrophotometer at 545 nm. Mixtures without the coupling reagent served as a control. The standard PABA solution was made up as described by Howitz (7). ° Both culture media were obtained from Grand Island Biological Company, New York, USA. b Sigma Chemical Company, St. Louis, MO, USA. 4191 -423- 424 C. R. BROCKELMAN & P. TAN-ARIYA Response to sulfadoxine The response of the parasites to sulfadoxine was assessed by a Petri dish test, as described by Trager et al. (8), and by a vial test (9). Parasites were grown in RP in 60-mm Petri dishes for 4 days, when a4% para- sitaemia was reached, then pooled and washed repeatedly in WM. A total of 64 subcultures were made in 35-mm Petri dishes, 32 of which received WM medium and 32 RP. Cultures were maintained without drug for 2 days and then exposed for 2 days to sulfadoxine dissolved in the appropriate medium at a concentration of 0, 0.5, l, 2, 4, 6, 8, or 10 mmol/litre (prepared from a 1 mol/litre solution of sulfa- doxine).c Parasite counts per 10 000 erythrocytes were made at the beginning of the experiment, before exposure, and after exposure. Parasite population rates of change during the 2 days of drug treatment were expressed as the common logarithm of the para- site multiplication factor (9). To study the incorporation of [14C]-sulfadoxine, 4- day old cultures were centrifuged at 250 g to concen- trate the erythrocytes harbouring ring forms. The dark top layer was discarded and the remaining packed cells were subcultured in RP or WM. An initial parasitaemia of 1 G increased to 10% (80Gb trophozoites) within 3 days in RP or WM, and the culture was then exposed to [14CJ-sulfadoxine (7.5 MBq/litre (0.2 mCi/litre) prepared from a 1 mol/litre ['4CJ-sulfadoxine solution with a radio- activity of 37 x 103 MBq/litrec) in WM or RP in a candle jar for 24 h at 37 'C. The culture medium was removed and the erythrocytes were washed three times in cold phosphate-buffered saline at pH 7.2. The packed cells were then resuspended with an equal volume of phosphate-buffered saline to make a 50% cell suspension. A 25-pu aliquot was placed on a fibre- glass filter, dried at 37 'C overnight, then dropped in dioxane scintillation cocktail and counted on a Beck- man LS liquid scintillation counter. The number of erythrocytes per ml was estimated from a further 25- ,d portion of cell suspension in a Neubauer chamber. Normal non-infected cells were cultured in either RP or WM and tested in an identical manner. Statistical analysis All parasite multiplication rates were tested by 2- way analysis of variance for the effects of drug concentration and culture medium. Incorporation of [14C1-sulfadoxine into infected and non-infected erythrocytes cultured in RP or WM was analysed in a single classification ANOVA (10). ' Hoffmann-La Roche, Basel, Switzerland. RESULTS The sensitivity of FCM2 and FCM5 to sulfadoxine at all concentrations was much higher in Waymouth culture medium than in RP (Fig. 1). There was no growth of the parasite population of either strain in WM medium containing only 0.5 mmol of sulfadoxine per litre, whereas, in RP, the parasite growth at this concentration was similar to that of the control group. A sulfadoxine concentration of 6.4 mmol/litre was required for 50% inhibition of FCM5 in RP. Waymouth medium (without human serum) was found not to contain PABA, but 0.025 mg of PABA per litre was detected in complete WM containing 100 ml of human serum per litre. The concentration of PABA in RP was found to be 1.025 mg/litre, i.e., 41 times higher than that in WM. A further experi- ment, shown in Fig. 2, demonstrated that the FCM5 strain was inhibited in WM by 0.2 mmol/litre of sul- fadoxine, but was unaffected in RP medium at this concentration (see Table 1, P < 0.001). The mean parasite multiplication rate in WM was only 0.48, a value achieved in RP only at a drug concentration of 6.4 mmol/litre. Thus, 32 times more sulfadoxine was required for 50% inhibition in the presence of a 41- fold higher concentration of PABA. Furthermore, the addition of 1 mg of PABA per litre of WM decreased the drug response of the parasite to the level seen in RP (Fig. 2). The rate of incorporation of sulfadoxine in P.falciparum culture is shown in Table 2. The amount of drug incorporated into the non-infected erythrocytes was 15.9 times higher in WM (11 003 cpm) than in RP (692 cpm). The difference was even greater in parasite cultures in which 10% of 2 c0 E 0) __ -I o 2 4 6 8 10 Sulfodoxine (mmol / I medium) Fig. 1. Response of P. falciparum strains FCM2 and FCM5 to sulfadoxine in the Petri-dish test, showing mean parasite multiplication rate and standard deviation. A- -A , FCM2 in RP; A- -A, FCM2 in WM; 0-0 FCM5 in RP; A-6 , FCM5 in WM. SULFADOXINE SENSITIVITY OF P. FALCIPARUM the erythrocytes were infected with trophozoites of P.falciparum; the counts were 62 900 cpm in WM and 2800 in RP (a 22.5-fold difference). DISCUSSION Our experimental results indicate that the apparent in vitro susceptibility of P.falciparum to sulfadoxine is influenced by the culture medium. RPMI was used o 0'1 't 0.4 04 1 54t *14 lS in the first successful continuous culture of P.falci- parum by Trager & Jensen (1) and has since been Sulfadoxine (rnmol /I medium) adopted internationally. Because of its PABA Response of P. falciparum strain FCM5 to sulfa- centration of 1 mg per litre, this synthetic medium showing mean parasite multiplication rate and supports growth of malaria parasites in vitro (11). rd deviation. 0--C in RP medium;conen i However, the PABA concentration in RPMI is much */R in WM with a PABA concentration higher than the actual requirement and thus the!5 mg/litre. medium is unsuitable for the in vitro investigation of antimalarial drugs that act by inhibiting either the utilization of PABA or the biosynthesis of folate Analysis variance, using a single classifi- precursors of the parasites. In 1950, Thurston (12) ANOVA, of parasite growth rates in WM and RP with various concentrations of sulfadoxine showed that PABA antagonizes the action of sulfa-diazine against P. berghei in mice. The inhibition of Degrees P. berghei by a milk diet was demonstrated to be due of Mean F P to a PABA deficiency (13). The addition of human freedom square serum to the Waymouth formula medium provides sufficient PABA (250-300 jtg/litre of serum) for the)f variation growth of the parasite. Studies in our laboratory (un- m 1 633 938 134.62 < 0.001 published observations) have demonstrated that loxine Waymouth medium supplemented with human serum centration 3 221 125 46.96 < 0.001 is as good as RPMI in supporting growth and asexual ction 3 195050 41.42 < 0.001 multiplication of P.falciparum, as evidenced by glucose utilization, incorporation of [3HI-isoleucine, ubgroups 24 4 709 and the parasite multiplication rate. The observed sensitivity to sulfadoxine is thus not an artefact of an inappropriate culture medium. The kinetics of sulfadoxine incorporation into infected cells is being 2. Incorporation of [1'4Cl-sulfadoxine into 500 x studied in our laboratory. ythrocytes after 24 h incubation in WM or RP The test system reported here is being applied to the in vitro microtechnique (14) to assess sulfadoxine sensitivity using blood from patients in various a- Incorporation of [14Cl-sulfadoxine (cpm x 1 0-3) endemic areas. It is hoped to obtain information on mia the sensitivity of P.falciparum in Thailand to sulfa RP WM P drugs and to determine their minimum inhibitory concentrations. The in vitro cultivation of parasites 692±14.32 11 003 ± 930 < 0.001 using Waymouth formula medium will also provide a 2800± 235.51 62 900±13 958 <0.001 system for investigation of the pathway of dihydro- ._______________________________________________ folate biosynthesis in malaria parasites. ACKNOWLEDGEMENTS This study received financial support from the UNDP/World Bank/WHO Special Vrogramme for Research and Training in Tropical Diseases and from the National Research Council of Thailand. We thank Dr S. Ngamphnom of the Department of Infectious Disease Control, Malaria Control Centre, Chantaburi, for parasite materials, and Dr R. Lasserre of the Roche Far East Research Foundation and F. Hoffmann-La Roche & Co. for drugs used in this study. 1 - 0.O - 9- 0.4 tn 0 0. -0.4 Fig. 2. doxine, standai in WM of 1.02 Table 1 cation, media, Source c Mediu Sulfad con, Intera Within s Table 107 er mediui Par, sitaei C ic 425 426 C. R. BROCKELMAN & P. TAN-ARIYA RESUME PLASMODIUM FALCIPARUM EN CULTURE CONTINUE: UN NOUVEAU MILIEU POUR L'EPREUVE DE SENSIBILITE A LA SULFADOXINE IN VITRO Pour d6terminer la reponse a la sulfadoxine de Plasmo- dium fakciparum de Thailande, on a utilise un systeme in vitro et la methode employant des boites de Petri et une cloche a bougie. Les souches thailandaises FCM2 et FCM5 de P.falciparum ont ete cultiv&es en milieux RPMI 1640 et Waymouth contenant 25 mmol de HEPES et 2 g de NaHCO3 par litre, additionn6s de 100 ml de s6rum humain par litre (milieux d6signes respectivement comme RP et WM). Le dosage de I'acide amino-4 benzoique (PABA) dans les deux milieux a r6vele des concentrations de 1,025 mg/litre dans RP et de 0,025 mg/litre dans WM. Les r6ponses des deux isolements de P.falciparum a la sulfa- doxine ont 6t6 tres faibles dans RP, oi il fallait une concen- tration de medicament de 6,4 mmol/litre pour obtenir une inhibition de 50% de la croissance des parasites, alors qu'il n'y avait plus aucune croissance dans le milieu WM A une concentration de 0,5 mmol de sulfadoxine par litre. D'autres experiences ont confirme une inhibition hautement significative dans le milieu WM par une concentration du m6dicament aussi faible que 0,2 mmol/litre. La difference entre les resultats observ6s dans les milieux RP et WM a et attribuee A une comp6tition entre le PABA et la sulfadoxine, analogue du PABA. Cela a ete confirm6 par le fait que 1) la croissance des parasites n'6tait que faiblement modifi&e par la sulfadoxine en milieu WM auquel du PABA supplementaire avait &e ajout6, et que 2) la sulfadoxine marqu6e par 14C etait incorpor6e dans les erythrocytes beaucoup plus rapidement en milieuWM qu'en milieu RP. REFERENCES 1. TRAGER, W. & JENSEN, J. B. Science, 193: 673 (1976). 2. RICHARDS, W. H. G. & MAPLES, B. K. Annals of tropical medicine and parasitology, 73: 99 (1979). 3. BROCKELMAN, C. R. & TAN-ARIYA, P. Report given at the 10th International Congress of Tropical Medicine, Manila, The Philippines, November 1980. 4. CHEN, P. ET AL. Southeast Asia journal of tropical medicine and public health, 11: 435 (1980). 5. Catalog ofsynthetic media, International edition, New York, Grand Island Biological Company, 1977, p.141. 6. SOMOGYI, M. Journal of biological chemistry, 86: 655 (1930). 7. HOWITZ, W., ED. Official methods of analysis of Association of Official Analytical Chemists. 1975, p.778. 8. TRAGER, W. ET AL. FEBS letters, 85: 264 (1978). 9. BROCKELMAN, C. R. ET AL. Bulletin ofthe WorldHealth Organization, 59: 249 (1981). 10. SOKAL, R. R. & ROHLF, F. J. Biometry, San Fran- cisco, W. H. Freeman, 1969. 11. FERONE, R. Bulletin of the World Health Organization, 55: 291 (1977). 12. THURSTON, J. P. Lancet, 2: 438 (1950). 13. KRETSCHMAR, W. Zeitschrift fur Tropenmedizin und Parasitologie, 17: 368 (1966). 14. RIECKMANN, K. H. ET AL. Lancet, 1: 22 (1978).