In vitro cultivation of malaria parasites

Abstract

Interest in cultivation of the causative agent of human malaria must have followed soon after Laveran's historic observation of the living parasite in a fresh blood specimen. The first recorded report of in vitro culture of Plasmodiumfalciparun~ was made in 1912 by Bass and Johns (2). Since that time there have been many attempts to maintain the erythrocytic cycle of the parasite in cultures, primarily with avian, simian or murine species of Plasmodiunt. It was not until 1976 that the final essential elements of a successful method for the continuous cultivation of Plasmodium falciparum in human erythrocytes were discovered and reported (4, 10). The new technique primarily differs from the many earlier reported attempts in that it employs dilute cultures with low initial parasite densities and an atmosphere with reduced oxygen tension (4, 10). Successful in vitro cultivation of P. falciparum and other species of Plasmodium has now been reported by many investigators in various laboratories. The culture medium most commonly employed consists of buffered RPMI 1640 supplemented with 10-15070 human serum or plasma (10). Frequent dilution with a fresh red blood cell suspension and daily changes of the culture medium are essential. In this way a continuous but asynchronous intraerythrocytic cycle can be maintained indefinitely with parasites infecting 10070 or more of the erythrocytes. Several isolates of P. falciparum with varying susceptibility to antimalarial agents have now been successfully adapted to continuous in vitro culture (3, 5), providing an unparalleled resource for research on the biology, metabolism, immunology and chemotherapy of human malaria. The most immediate impact of this new technology has been in the field of antimalarial chemotherapy. A resurgence of interest in antimalarial drug research was triggered in the early 1960s by the initial appearance of chloroquine-resistant falciparum malaria in Colombia. Resistance to other antimalarial drugs such as pyrimethamine and chloroguanide was also then widespread. By the early 1970s chloroquine-resistant isolates of P. falciparunl had been reported throughout Southwest Asia, Central and South America and in India. Recently, the first chloroquineresistant isolate on the African continent was reported from Kenya (6). The treatment and prophylaxis of falciparum malaria has again become a complex and difficult problem (1). It is of great epidemiologic interest and therapeutically helpful to rapidly determine the susceptibility of a strain of P. falciparum to chloroquine and other antimalarial drugs. The use of short-term cultures of P. falciparum directly from clinical specimens to evaluate chloroquine susceptibility was described by Rieckmann in 1971 (8). This simple but extremely useful method was a modification of the technique originally described by B.ass and Johns (2). In a defibrinated whole blood specimen supplemented with glucose and incubated at 38°C, intraerythrocytic maturation of the parasites from trophozoites to multinucleated schizonts occurs in vitro within 24 hours. Chloroquine (10 ng/ml) will inhibit this maturation if the isolate is susceptible. Resistant isolates will undergo normal maturation even at 5-10 times higher concentrations of the drug. This in vitro method is useful for epidemiologic studies and is the World Health Organization standard test to determine chloroquine susceptibility. Use of the method is limited by its requirement for blood specimens that contain parasites at the proper stage of development for initiation of the culture. Because of poor maturation of the parasites, the results are sometimes unreliable when the parasite density in the specimen is high. While other 4-aminoquinolines and quinine could also be evaluated readily by this technique (8), other drugs such as pyrimethamine, which requires more than a single intraerythrocytic cycle to exert a suppressive effect, could not. Rieckmann et al. (9) more recently described a modification of the earlier method that has proved more reliable. The new method uses microtitration plates that are prepared in advance by the addition of solutions of chloroquine to selected wells of the plates, which are then dried and stored. Small