Decrease in susceptibility of Plasmodium falciparum to mefloquine in continuous culture*

Bulletin of the World Health Organization, 59 (2): 249-252 (1981) Decrease in susceptibility ofPlasmodiumfalciparum to mefloquine in continuous culture* C. R. BROCKELMAN,1 S. MONKOLKEHA,2 & P. TANARIYA3 Chloroquine-resistant P. falciparum, line FCK from Thailand, was tested for susceptibility to mefloquine in continuous culture. The drug resulted in SO%o schizont inhibition at the level of 8. 0 nmol/ml of culture when testedfor thefirst time. After three months of discontinuous exposure to mefloquine at increasing levels, the sensitivity of the parasite decreased. A concentration as high as 128 nmol/ml eventually was requiredforS5o% inhibition ofgrowth. The occurrence of chloroquine resistance in Plas- modiumfakciparum in Thailand has increased rapidly since it was first reported by Harinasuta in 1961 (1). More recent data from this country has shown that about 997o of isolates are resistant to chloroquine (2). Chemoprophylaxis and treatment of chloroquine- resistant P.falciparum has been possible with a combination of pyrimethamine and sulfadoxine (3). However, it has been reported recently that higher doses of this drug are required for treatment, and the cure rates are lower than was previously the case.a This indicates that P.falciparum in Thailand is also developing resistance to this drug combination. Synthesis and screening of 60 000 compounds by the US Army has given rise to one promising drug, mefloquine. It is an analogue of quinine and of the experimental drug WR 30090, which was used suc- cessfully to treat chloroquine-resistant falciparum malaria in Viet Nam (4). Single-dose treatment with mefloquine gave satisfactory results and it was also found that this drug actively suppresses both falci- parum and vivax malaria. It is well tolerated and produces no side effects at a dose as high as 500 mg/kg of body weight (5). In vitro studies of responsiveness of chloroquine-resistant P.falciparum to mefloquine showed that concentrations of 1.0 nmol per ml of blood could readily inhibit development of schizonts in all of the cultures tested in Thailand (6). Using the Petri dish continuous culture system developed by Jensen & Trager (7), we have observed that a concentration as low as 0.05 nmol/ml of medium inhibited the growth of about half the popu- * From the Department of Microbiology, Faculty of Science, Mahidol University, Rama VI Rd, Bangkok 4, Thailand. IAssociate Professor. 2 Research Associate. 3Predoctoral Fellow. a Williams, R. Unpublished data, March 1980. lation of P.falciparum. Healthy ring forms would develop into schizonts if the mefloquine was removed and fresh erythrocytes were added to the culture. These surviving parasites appeared to tolerate meflo- quine when treatment was repeated. It was therefore of interest to investigate whether or not selection for resistance to mefloquine was occurring. MATERIALS AND METHODS P.falciparum line FCK was isolated from a patient in Kanchanaburi, west Thailand, in January 1979. Tests for response to chloroquine (8) revealed that the isolate was resistant to chloroquine at the level of 1 nmol/ml of blood. The parasites were maintained in continuous culture by the Petri dish cultivation method described by Jensen & Trager (7). They were subcultured regularly every 4 days in erythrocytes group 0, the percentage parasitaemia on day 0 always being 0.2%. The culture medium consisted of RPMI 1640, with the addition of 50 g of NaHCO3 and 100 ml of heat-inactivated (56 IC, 30 min) human serum, group 0, per litre. This medium is referred to below as control medium. Mefloquine hydrochloride was obtained as a 1 mmol/litre sterile solution from the World Health Organization, Geneva, Switzerland. The stock sol- ution was diluted with sterile, deionized water to 100 times the required concentration. A working solution was made by further dilution with RPMI + NaHCO3, but without serum. The final required concentrations were obtained by adding different volumes of the working solution to the control medium. Precaution was taken that not more than 0.1 ml of the working solution was used per 10 ml of the control medium in order to maintain the human serum: RPMI + NaHCO3 ratio within a narrow range. 4052 -249- C. R. BROCKELMAN ET AL. The parasite responses to mefloquine were tested in replicated glass-covered vials (2 cm x 3 cm), each con- taining 0.1 ml of erythrocytes, with parasitaemia of 0.3%, suspended in 0.9 ml of control medium. We replaced the control medium with the test medium (control medium + mefloquine) on the second day of subculture. Generally, the cultures were exposed to the drug for 48 hours. Giemsa-stained thin blood films were made before drug treatment and 48 hours after- wards. Four replicates were used for each drug level and for the controls. To produce drug tolerance, the cultures were exposed to mefloquine at a concentration of 0.04 nmol/ml for 48 hours. Cultures that showed healthy ring forms were subcultured with non-infected, fresh erythrocytes and control medium and maintained without drug for several cycles. Cultures were treated again with mefloquine at a 2-4 times higher concentration. After three months the mefloquine-treated line (designated FCL-R) was unaffected by this antima- larial at a concentration of 32 nmol/mol, and its sensi- tivity to mefloquine was then compared with that of the original line (FCK-S). Mefloquine was added to cultures of both lines at concentrations of 8, 16, 32, 64 and 128 nmol/ml. Evaluation of mefloquine inhibition was done by direct counting of parasites in thin blood films made before drug treatment (day 2) and 48 hours after treat- ment (day 4). The multiplication factor (MF) of the parasites is N(day 4)/N(day 2) where Nis the number of parasites/10 000 red blood cells on the day indi- cated. Because the multiplication is exponential, a log- arithmic transformation is used: log MF = log N (day 4) - logN (day 2) The 11 groups of rates (log MF), excluding the group with all zeros, were found to have homogenous vari- ances (Bartlett's test: ,2 = 39.09, 0.5 >P>0. 1) and so two-way ANOVA was carried out to test the effects of drug concentration and culture line. The highest con- centration was omitted from the analysis because of the constraint in group variance. RESULTS The in vitro responses to mefloquine of the FCK line, before (FCK-S) and after production of drug tolerance (FCK-R) are summarized in Fig. 1. The sensitive line, which has never been exposed to meflo- quine, showed a steady drop in the rate of population increase with increase in the concentration of the drug. Complete inhibition occurred at a concentration of 128 nmol/ml. The FCK-R line was not inhibited by any of the 0.5- 0 c0 .-1C 0 .6-0 U ._ E V a 0.4 l- 0.31 0.2 H 0.I H x If, 0 " 8 16 32 Dose of mefloquine in nmol/ml (logarithmic scale) Fig. 1. Response of the two lines of P. falciparum to meflo- quine in continuous cultures. Parasite population increases during 48 h of drug treatment are presented as the common logarithm of the multiplication factor (MF). Closed circles represent log MF of FCK-R, the line that was repeatedly exposed to mefloquine; crosses represent the FCK-S line that was not exposed to mefloquine. The lines connect the means at each dose level. lower concentrations (8-64 nmol/ml). Only at 128 nmol/ml was inhibition first evident. The mean multi- plication factor with two days of treatment at that concentration was 0.089 which was higher than that of the sensitive line at 128 nmol/ml. Table 1 shows a significant effect difference between parasite lines (F1, 30 = 21.64, P<0.001), indicating that line FCK-R had reduced susceptibility to mefloquine. The change in the sensitivity of the FCK-R line is indicated by the significant interaction mean square. Only at the highest concentration was there a marked depression in multiplication. Table 1. Analysis of variance of population growth rates (log multiplication factor) of P. fa/ciparum lines FCK-R and FCK-S when treated with mefloquine at various levels Source of variation df Mean square Line FCK-R versus FCK-S Mefloquine concentration Interaction Error F P 1 104 448 21.64 0.001 4 101 44 4 140 673 30 4 827 21.02 0.001 29.14 0.001 Total 39 250 0.6 r x 0 SUSCEPTIBILITY OF P. FALCIPARUM TO MEFLOQUINE DISCUSSION Short-term in-vitro tests of the sensitivity of chloroquine-resistant P. falciparum to mefloquine have shown, in most cases, that mefloquine is fully effective in inhibiting the parasite's growth. Rieck- mann (6) reported that at 1.0 nmol/ml of blood no schizonts were observed in any of his 15 test cultures. This was confirmed by Lopez Antuftano & Werns- dorfer (9), who obtained 99.9%o schizont inhibition by mefloquine at a level as low as 2 pmol/5 pl of blood (0.4 nmol/ml). Our results obtained with continuous cultures also showed that the chloroquine-resistant P. falciparum line FCK was very susceptible to mefloquine when it was exposed to this drug for the first time. However, we found that the surviving ring stages could be re- vived and would develop into schizonts. This phenom- enon is comparable to the in vivo situation where drug treatment is terminated and medical follow-up ceases. In order to imitate this drug abuse situation, which may lead to tolerance or resistance to antimalarials, we did not expose P.falciparum cultures to the drug continuously as specified in the experimental design of Nguyen-Dinh & Trager (10). Recrudescence occurred, with the chance that the surviving parasites had become resistant to the drug. The misuse of chloroquine and the pyrimethamine- sulfadoxine combination by local people has given rise to resistance to both drugs. In consequence, we are concerned that when mefloquine is commercially available, it too might soon become ineffective. Our results showed that the "misuse" of mefloquine in culture, i.e., at a concentration lower than prescribed and with early termination of treatment, caused a decrease in sensitivity to mefloquine within three months. ACKNOWLEDGEMENTS This study was supported by the Thai National Research Council and in part, by the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases. We are also indebted to Dr S. Thaitong for the materials to initiate the cultures, Dr W. H. Wernsdorfer for supplying the mefloquine used in the study, and Dr W. Y. Brockelman for his advice with the statistical analysis. RESUME DIMINUTION DE LA SENSIBILITt A LA MtFLOQUINE CHEZ PLASMODIUM FALCIPARUM EN CULTURE CONTINUE Les difficultes qui sont rencontrees dans le traitement du paludisme A P. falciparum en Thailande sont attribu&es A une pharmacoresistance des parasites. Des donn&es recentes obtenues par application de la methode de Rieckmann ont revel que 990%o des isolements &aient resistants A la chloro- quine. En outre, on a signale que la chimioprophylaxie et le traitement au moyen de la pyrimethamine et de la sulfa- doxine deviennent moins satisfaisants. L'introduction d'un medicament nouvellement synth&e tise, le WR 30090 (mefloquine), qui est un analogue de la quinine, a permis un traitement efficace par une dose unique. Cependant nous avons observe que lorsqu'on eprouvait de la mefloquine contre P. falciparum etabli en culture continue, il y avait un petit nombre de jeunes parasites qui semblaient tolerer ce medicament. Il importait donc de rechercher si une selection de parasites resistants A la mefloquine se produisait. P. falciparum utilise dans la presente etude a e isole d'un malade dans la province de Kanchanaburi, A l'ouest de la Thailande, en janvier 1979. Les epreuves destin&es A deter- miner les reponses de cette souche A la chloroquine ont montre qu'elle etait resistante A la concentration de 1 nmol/ml de sang. Le parasite a e maintenu en culture continue dans du milieu RPMI 1640 avec tampon HEPES, additionne de 1007 de s&rum humain. Le chlorhydrate de mefloquine obtenu aupres de l'OMS a e ajoute aux cultures A la concentration initiale de 40 pmol/ml de milieu. Les cultures ont e soumises A la pression du medicament pendant 48 heures puis transf&ers et remises en culture avec des erythrocytes non infectes en suspension dans du milieu exempt de medicament. La culture a de nouveau e trait&e par la mefloquine A une concentration augmentant chaque semaine. Apres trois mois, la lign&e trait& par la mefloquine (FCK-R) n'etait plus affect& par cet antipaludique A une concentration de 32 nmol. Sa sensibilite A la mefloquine a alors et comparee A celle de la lignee originelle (FCK-S) aux concentrations de 8, 16, 32, 64 et 128 nmol. L'evaluation a e faite par l'analyse de la variance du facteur de multiplication (FM) du parasite [log FM = logN (jour 4)-log N (jour 2)]. La souche sensible qui n'avait jamais e exposee A la mefloquine a presente un abaisse- ment regulier du taux d'accroissement de la population, correspondant A la concentration du medicament. La lignee FCK-R n'etait inhibee par aucune des concen- trations les plus faibles (8-64 nmol). Ce n'est qu'A 128 nmol que le medicament manifestait un effet inhibiteur. I1 y avait une difference significative de l'effet selon la lignee de parasites (F, 30 = 21,64, P<0,001), indiquant qu'une 251 252 C. R. BROCKELMAN ET AL. lignee FCK-R avait une sensibilite reduite A la mefloquine. C'est seulement A la plus haute concentration qu'on obser- vait un flchissement marque de la multiplication. Les resultats de nos experiences ont clairement montre que la ((mauvaise utilisation>> de la mefloquine dans la culture, c'est-A-dire A une concentration plus basse que celle qui est prescrite, avec un arret precoce du traitement, entraine facilement une diminution de la sensibilite A la mefloquine dans les trois mois. REFERENCES 1. HARINASUTA, T. ET AL. In: Proceedings ofthe UNESCO First Regional Symposium on Scientific Knowledge of Tropical Parasites, Singapore, 1962, 1962, p. 148. 2. SUCHARIT, P. ET AL. Annals of tropical medicine and parasitology, 71: 401-405 (1977). 3. HALL, A. P. ET AL. British medical journal, 2: 15-17 (1975). 4. DOBERSTYN, E. B. ET AL. Bulletin of the World Health Organization, 57: 275-279 (1979). 5. CLYDE, D. F. ET AL. Antimicrobial agents and chemo- therapy, 9: 384-386 (1976). 6. RIECKMANN, K. H. Susceptibility of cultured parasites of Plasmodium falciparum to antimalarial drugs. In: The in vitro cultivation of the pathogens of tropical dis- eases. 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