Pyrimethamine sensitivity in Plasmodium falciparum: determination in vitro by a modified 48-hour test*

Bulletin of the World Health Organization, 58 (6): 909-912 (1980) Pyrimethamine sensitivity in Plasmodium falciparum: determination in vitro by a modified 48-hour test* P. NGUYEN-DINH1 & D. PAYNE 2 Four strains of Plasmodium falciparum recently isolated in culture were assessed in vitro for their response to pyrimethamine. A simple modified 48-hour test was used, which showed two strains to be sensitive to the drug in vitro, while the other two were resistant at a very high level. In the two strainsfor which relevant clinical information was available the in vivo response to pyrimethamine was corroborated by the in vitro findings. This modified 48-hour test is thus usefulfor determining patterns ofdrug sensitivity in laboratory-adapted strains, and would be a valuable asset if found to be equally applicable under field conditions. Strains of malaria parasites resistant to pyri- methamine have been reported in many parts of the world (1). Their occurrence restricts the use of this valuable antimalarial drug and has now assumed a new importance since pyrimethamine is increasingly used in drug combinations directed against chloro- quine-resistant Plasmodiumfalciparum (2). It is now possible, by continuous cultivation tech- niques (3), to maintain different strains of P.falciparum in the laboratory, and to test in vitro their response to various drugs (4), including pyrimethamine (5, 6). A modified 48-hour test that is much simpler than the previous methods was described recently (7) for the determination in vitro of chloroquine sensitivity in laboratory-adapted strains of P.falciparum. This modified 48-hour test has now been successfully used to assess the pyrimethamine sensitivity of four newly isolated strains of P.falciparum. MATERIALS AND METHODS Origins of the strains Three strains of P.falciparum were isolated in January-March 1980 from malaria cases imported into the United States of America. Blood samples from the patients were collected in heparinized tubes and sent to the Center for Disease Control (Atlanta, * Requests for reprints should be addressed to Mr D. Payne. Visiting Associate, Bureau of Tropical Diseases, Center for Disease Control, Public Health Service, Department of Health and Human Services, Atlanta, GA 30333, USA. 2 Technical Officer, Research and Technical Intelligence, Malaria Action Programme, World Health Organization, 1211 Geneva 27, Switzerland. Georgia) where they arrived within 10 hours. The samples were used to establish the parasites in culture by the standard Petri dish/candle jar method (8, 9). A fourth strain was isolated and started in culture during field studies in Choluteca, Honduras, in January 1980, before being hand-carried to Atlanta, where it was returned to culture after a total transportation period of 36 hours. In two of the strains the clinical history of the patient provided information on the in vivo response of the parasites to pyrimethamine. Strain Africa- I/CDC (clinically pyrimethamine-resistant) was isolated from a 57-year old white female who acquired P.falciparum malaria during a trans-African safari while under prophylaxis with 25 mg ofpyrimethamine weekly. Strain Sierra Leone-l/CDC (clinically pyri- methamine-sensitive) was isolated from a 19-year-old Sierra Leonean female in whomP.falciparum malaria was diagnosed 3 weeks after her arrival in the USA; the blood sample was collected before the patient was successfully treated with 50 mg of pyrimethamine daily for 2 days. The two remaining strains were Kenya-2/CDC, from a case of P.falciparum malaria imported from Kenya, and Honduras-l/CDC, from a patient seen during an outbreak of urban malaria in Choluteca, Honduras. No information was available on the in vivo pyrimethamine sensitivity of the parasites, since other antimalarials were used to treat the patients. Test method The modified 48-hour test described earlier (7) was adapted for use with pyrimethamine as follows. The starting material was prepared by washing uninfected O+ erythrocytes, resuspending them to 4017 -909 910 P. NGUYEN-DINH & D. PAYNE 50% in normal medium (RPMI 1640 mediuma supplemented with HEPES buffer (30 mmol/litre) and 100 ml of O+ serum per litre), and seeding them with parasites derived from continuous culture lines. The starting parasitaemias thus obtained varied between 10 and 70 parasites per 104 erythrocytes. Aliquots of 20 Ml of this starting material were distributed into flat-bottomed 16-mm wells contain- ing 0.5 ml of either normal medium or medium with various concentrations of pyrimethamine (0.3, 0.1, 0.03, 0.01, and 0.003 Mmol/litre, prepared from a 100 Mmol/litre aqueous stock solution of the drug). The final erythrocyte suspension was thus 2070. After shaking to ensure resuspension and uniform resettling of the erythrocytes, the trays were placed in a candle jar and allowed to incubate for 48 hours at 37 'C. After 24 hours the cells were resuspended by agitation of the trays and the gas phase in the candle jar was regenerated. Table 1. Response (parasite count per 104 erythrocytes) of strain Sierra Leone-i /CDC to pyrimethamine in the modi- fied 48-hour test Concentration of pyri- methaminea Gameto- Tropho- Total (g.mol/litre) cytes Rings zoites S =2Nb S>2NC count Time 0 0 9 2 1 3 15 At 48 h: Control 0 78 19 6 6 109 1 79 18 8 16 122 0.3 1 0 5 0 0 6 0 0 1 0 0 1 0.1 3 0 4 1 0 8 1 0 8 0 0 9 0.03 1 3 7 3 5 19 0 4 3 0 4 11 0.01 0 37 10 6 10 63 1 29 16 12 14 72 0.003 1 60 19 2 22 104 0 90 4 5 13 112 8 Duplicate wells were used for each concentration of pyri- methamine. b Schizonts with two nuclei. c Schizonts with three or more nuclei. a GIBCO Laboratories, Grand Island, New York, USA. (Use of trade names or commercial sources does not constitute endorsement by the Public Health Service of the US Department of Health and Human Services.) Parasite counts were made on Giemsa-stained thin smears taken at the beginning and end of the experi- ment. Both normal and abnormal parasites were included in the count. RESULTS The 48-hour test distinguished two patterns of response to pyrimethamine. 1. Strains Sierra Leone-I and Kenya-2 were found to be pyrimethamine-sensitive in a series of experi- ments which yielded consistently similar results. In both Sierra Leone-l (5 experiments) and Kenya-2 (3 experiments), complete inhibition of the parasite growth was observed in medium containing 0.03 pmol/litre of pyrimethamine. At this drug concen- tration no increase in parasitaemia occurred over 48 hours, and most of the parasites present were abnormal late trophozoites and schizonts, with only rare rings observed. No clear-cut effect on the gameto- cytes was apparent. Results from a typical experiment with Sierra Leone-I are shown in Table 1, and rep- resentative curves for both Sierra Leone-I and Kenya-2 are illustrated in Fig. 1. 2. Strains Africa-I and Honduras-I were found to be pyrimethamine-resistant. In both Africa-i (5 experiments) and Honduras-i (4 experiments), no inhibition was observed up to the maximal level of 0.3 pmol/litre (Table 2 and Fig. 1). Additional experi- ments using higher concentrations of the drug showed increases in parasitaemia in medium containing up to 3 pmol/litre of pyrimethamine, with absence of growth occurring only at 10 Amol/litre. 200 a:\ 0 ~~~~~~~~~~~~~Honduras-i 1)00 0 o \<_ Africa-1 T=0 Sierra Leone-i eL ~ tKenya-2 0 0.003 0.01 0.03 0.1 0.3 PYRIMETHAMINE (Pmo/lL medium) Fig. 1. Pyrimethamine response curves of strains Africa-1/ CDC, Honduras-i /CDC, Sierra Leone-1 /CDC, and Kenya- 2/CDC, in the modified 48-hour test. Each point represents the mean of two duplicate wells. The inset (T = 0) indicates the starting levels of parasitaemia. PYRIMETHAMINE SENSITIVITY IN P. FALCIPARUM Table 2. Response (parasite count per 104 erythrocytes) of strain Africa 1 /CDC to pyrimethamine in the modified 48-hour test Concentration of pyri- methaminea Gameto- Tropho- Total (ylmol/litre) cytes Rings zoites S =2Nb S>2NC count Time 0 0 27 1 5 0 4 46 At 48 h: Control 2 38 62 9 9 120 3 47 45 3 10 108 0.3 3 52 56 3 18 132 6 30 49 7 10 102 0.1 6 29 55 6 5 101 3 22 53 7 4 89 0.03 7 28 61 12 4 112 2 20 49 4 8 83 0.01 3 33 48 5 12 101 1 18 65 5 5 94 0.003 4 39 58 4 10 115 1 32 53 4 9 99 a Duplicate wells were used methamine. b Schizonts with two nuclei. for each concentration of pyri- c Schizonts with three or more nuclei. DISCUSSION This work confirms the value of the 48-hour test for assessing in vitro the drug sensitivity of laboratory- adapted strains of P.falciparum. Two different patterns of response to pyrimethamine were observed. Resistance to pyrimethamine, when present, was marked, the inhibition of the resistant strains occur- ring only at drug concentrations much higher than those required for the sensitive strains. This confirms observations made previously by Desjardins et al. (5) using a different in vitro method. In the two cases where a clinical history made avail- able the relevant information, a good correlation was found between the in vivo and in vitro responses of the parasites to pyrimethamine. The 48-hour duration of the test resulted in the parasites being exposed to the drug for a full cycle of asexual multiplication. This made it possible to study the effects of the drug on the morphology of all the parasite stages. Pyrimethamine appeared to act mainly on the late trophozoite and schizont stages, as found by Gutteridge & Trigg (10) in earlier in vitro studies using P. knowlesi. In the present study the decrease in the number of ring stages resulted from the inability of the abnormal schizonts to mature and release viable merozoites, and constituted a valid indicator of inhibition by pyrimethamine. The apparent absence of any effect of the drug on gameto- cytes was in accordance with the earlier in vivo observations by Burgess & Young (11). The possibility of adapting this simple test to field conditions needs to be explored, since a field method for the in vitro detection of pyrimethamine resistance is not currently available. The recent spread ofchloro- quine resistance to Africa (12) will result in a greater use of alternative drugs, such as combinations includ- ing pyrimethamine. Thus, a field test which could be used with antimalarials of diverse modes of action would be very valuable. ACKNOWLEDGEMENTS We thank Dr C. C. Campbell for his participation in the isolation and establishment in culture of the strains studied, and Dr R. E. Desjardins, Wellcome Research Laboratories, for providing the pyrimethamine powder. This work was supported by a grant from the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases. RESUME SENSIBILITE DE PLASMODIUMFALCIPARUMA LA PYRIMETHAMINE: DtTERMINATION IN VITRO AU MOYEN D'UNE CPREUVE MODIFItE DE 48 HEURES Une epreuve modifiee de 48 heures a e employee pour determiner in vitro la sensibilite A la pyrimethamine de 4 souches de Plasmodium falciparum recemment isol6es et maintenues en culture continue. On a reparti dans des cupules A fond plat de 16 mm des fractions de 0,5 ml soit de milieu normal (RPMI 1640 additionne de 30 mmol/litre de tampon Hepes et de 10% de serum humain), soit de milieu contenant diverses concentrations de pyrimethamine. Puis, 911 912 P. NGUYEN-DINH & D. PAYNE on y a ajoute des fractions de 20 ;I d'une suspension a 50% d'erythrocytes ifect6s par P.falciparum (parasitemie: 0,1 A 0,7%). Apres incubation dans une cloche a bougie & 37°C pendant 48 heures, I'accroissement de la parasitemie a e mesure pour evaluer I'action inhibitrice du m6dicament. Deux des souches (Sierra Leone-l/CDC et Kenya-2/CDC) se sont montrees sensibles a la pyrimethamine in vitro, la parasitemie n'ayant pas augmente en presence de 0,03 amol/ litre. Les 2 autres souches (Africa-l/CDC et Honduras- 1/CDC) etaient hautement resistantes, l'inhibition ne s'etant exercee qu'a une concentration de lOmol/litre. Pour les souches Sierra Leone-I et Africa-i, on disposait de donnees cliniques qui ont permis de constater une bonne correlation entre les reponses A la pyrimethamine in vivo et in vitro. La simplicite d'execution de cette epreuve la place en bonne position pour des essais d'application sur le terrain, d'autant plus que la pyrimethamine fait de plus en plus frequemment partie des associations de medicaments utilisees contre le paludisme resistant A la chloroquine. REFERENCES 1. PETERS, W. 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