Parasite cultivation in relation to research on the chemotherapy of malaria.

Attempts to develop techniques for the continuous in vitro cultivation of the malaria parasite have not yet proved successful. It has not been possible to obtain the complete sporogonic development of the parasite in vitro although some progress was made with Plasmodium relictum and P. berghei. Exoerythrocytic stages of P. gallinaceum have been successfully cultivated in vitro in tissue explants and those of P. fallax have been grown in turkey primary embryo tissue cells. With the recent development of mammalian liver cell lines, prospects for the in vitro cultivation of exoerythrocytic stages of mammalian plasmodia are greatly improved. While it is still not possible to cultivate erythrocytic stages of plasmodia serially in vitro some species have been successfully grown through one asexual cycle. This progress has led to a number of applications of parasite cultivation to chemotherapeutic studies, to the testing of new antimalarial drugs, and especially to the testing of the susceptibility of P. falciparum to chloroquine. Cultivation technique is greatly improved by an appropriate choice of culture media. The addition of fresh red cells to the subculture system permits relatively high rates of invasion and multiplication of the parasite to be obtained. As well as its application in the screening and evaluation of antimalarial compounds, the in vitro cultivation technique is also very suitable for studying the entry mechanism of the parasite into red blood cells.     

Diagnosis of Plasmodium falciparum infection using a solid-phase radioimmunoassay for the detection of malaria antigens

A serodiagnostic test has been developed for the detection of Plasmodium falciparum in infected blood. Using parasite antigens and infected red blood cells from in vitro cultures of P. falciparum and malaria antibody from high-titre Gambian sera, parasites were detected in a solid-phase radioimmunoassay (RIA) that measured antibody-binding inhibition. Lysed RBC were incubated with labelled IgG purified from immune sera and were then placed in antigen-coated microtubes and incubated. The degree of inhibition of antibody binding in the tubes correlated with the level of parasitaemia in the test RBC and, using dilutions of RBC from in vitro cultures of P. falciparum, the test detected infection at a level of 8 parasites/10⁶ red blood cells. The test was applied to RBC from 100 healthy European blood donors and to samples of RBC from 500 Gambians from the up-country villages of Keneba and Manduar. More samples were positive by RIA than by microscopy and there was a highly significant degree of correlation between the RIA and microscopy results.     

Diagnosis of Plasmodium falciparum infection in man: detection of parasite antigens by ELISA

An ELISA method has been developed for the diagnosis of Plasmodium falciparum infection in man. Parasites from in vitro cultures of P. falciparum were used as source of antigen for the solid phase and the source of specific antibody was immune Gambian sera; binding of antibody in antigen-coated wells was registered by means of alkaline phosphatase-conjugated anti-human IgG. Parasites were detected on the basis of inhibition of antibody-binding. The test was applied to the detection of parasites in human red blood cells (RBC) from in vitro cultures of P. falciparum and in RBC from infected Gambians; RBC from 100 Geneva blood donors served as normal, uninfected controls. In titration experiments, the degree of antibody-binding inhibition correlated with the number of parasites in the test RBC. Parasites were detected at a level of 8 parasites/10⁶ RBC. Samples of RBC were tested from 126 Gambians with microscopically proven infection; significant antibody-binding inhibition was found in 86% of these cases, where parasitaemia ranged from 10 to 125 000/μl of blood. The presence of high-titre antibody in the test preparations was found to reduce the sensitivity of parasite detection in infected RBC from in vitro cultures mixed with equal volumes of different antibody-containing sera. The sensitivity was restored in most cases by recovering the RBC by centrifugation before testing. In a preliminary experiment, there was no significant difference in antibody-binding inhibition using fresh infected RBC and RBC dried on filter-paper and recovered by elution, although there was greater variation in the latter samples.     

Blood transfusion induced malaria in Iran

Blood transfusion plays the main rôle in induced malaria in Iran. Over 111 cases of transfusion malaria were recorded during the 10 years from 1963 to 1972. Seventy-three% of the species of plasmodia have been P. malariae and 27% P. vivax. 9 cases of transfusion induced quartan malaria in blood recipients have been studied. In 2 blood donors who were proved to be carriers of P. malariae by the fluorescent antibody test, scanty malaria parasites were detected in thick films made from blood concentrate obtained by centrifugation.     

Altered red cell membrane fluidity during schizogonic development of malarial parasites (Plasmodium falciparum and P. lophurae)

The plasma membranes of human or duckling erythrocytes infected with malarial parasites (Plasmodium falciparum and P. lophurae respectively) were stained by the fluorescent dye merocyanine 540 in the presence of serum. Unparasitized erythrocytes from infected ducklings or from in vitro cultures remained unstained in the presence of serum. Because merocyanine 540 has a greater affinity for fluid phased or disordered lipid bilayers the results suggest that upon infection of the red blood cell the erythrocyte plasma membrane becomes disordered or is increased in its fluidity. Such alterations of the host erythrocyte are probably due to parasite-induced modifications in the underlying spectrin network (required for lipid leaflet asymmetry) as well as changes in erythrocyte membrane lipid composition.     

Growth of protozoa in tissue culture. III. Trypanosoma cruzi

The technique used for cultivation of exoerythrocytic forms of P. gallinaceum has been applied to the growth of Trypanosoma cruzi also. A suspension of trypanosomes from the blood of an infected mouse was added to cultures of rat embryo. During the first 2 days great numbers of trypanosomes were present in the culture fluid, not particularly associated with the cells. Then they gradually became fewer until by about the 12th day they were rare or absent. Later small numbers reappeared in the culture fluid and persisted there until the end of the culture. Stained preparations made after. 6 days or more showed great numbers of intracellular parasites in all stages between the rounded leishmanoid form and the mature trypanosome form. The parasite occurred in cardiac muscle fibres, in macrophages and in elongated cells with processes (probably reticulo-endothelial cells). Cultures have been maintained for 59 days. As described by Meyer (1942) T. cruzi could also be grown in cultures from the brain of chick embryo.This technique offers favourable conditions for study of the intracellular development of T. cruzi and of the action of drugs upon it.     

Pf332-C231-reactive antibodies affect growth and development of intra-erythrocytic Plasmodium falciparum parasites

The Plasmodium falciparum antigen 332 (Pf332), is a megadalton parasite protein expressed at the surface of infected red cells during later stages of the parasite's developmental cycle. Antibodies to different parts of this antigen have been shown to inhibit parasite growth and adherence to host cells with or without ancillary cells. However, the mechanisms involved in these inhibitions remain largely unknown. We further analysed the activities of specific antibodies with regard to their specific mechanisms of action. For these analyses, affinity purified human antibodies against epitopes in the C-terminal fragment of Pf332 (Pf332-C231) were employed. All purified antibodies recognized Pf332-C231 both by immunofluorescence and ELISA. IgG was the main antibody isotype detected, although all sera investigated had varying proportions of IgG and IgM content. All the antibodies showed a capacity to inhibit parasite growth in P. falciparum cultures to different extents, mainly by acting on the more mature parasite stages. Morphological analysis revealed the antibody effects to be characterized by the presence of a high proportion of abnormal schizonts (15-30%) and pyknotic parasites. There was also an apparent antibody effect on the red cell integrity, as many developing parasites (up to 10% of trophozoites and schizonts) were extracellular. In some cases, the infected red cells appeared to be disintegrating/fading, staining paler than surrounding infected and uninfected cells. Antigen reversal of inhibition confirmed that these inhibitions were antigen specific. Furthermore, the growth of parasites after 22-42 h exposure to antibodies was investigated. Following the removal of antibody pressure, a decreased growth rate of these parasites was seen compared to that of control parasites. The present study confirms the potential of Pf332 as a target antigen for parasite neutralizing antibodies, and further indicates that epitopes within the C231 region of Pf332 should constitute important tools in the dissection of the role of Pf332 in the biology of the malaria parasite, as well as in the design of a malaria vaccine.     

Use of the in vitro microtechnique for the assessment of drug sensitivity of Plasmodium falciparum in Sennar, Sudan

In 1978, studies on the chloroquine sensitivity of Plasmodium falciparum were carried out in the district of Sennar, Sudan. The results of the in vivo tests showed parasites resistant at the RI level only, but the mean clearance time of trophozoites from the blood was higher than for strains found in many other areas of tropical Africa. The in vitro tests, using the microtechnique, indicated a lower sensitivity to chloroquine in the local P. falciparum isolates than in those of most other African countries. However, similar results have been reported from Ethiopia. The chloroquine sensitivity of P. falciparum from Sennar is close to the critical level of resistance. The in vitro microtechnique was also used to test for the sensitivity to Dabequin, 4-aminobenzo-quinoline, and was generally found to be a suitable and reproducible method, with a greater potential than the standard macro method. At parasite densities of over 100 000 asexual parasites per microlitre of blood the effect of a given concentration of chloroquine was related to the parasite density owing to the selective uptake of the compound by the parasitized cells.     

The interaction between sickle haemoglobin and the malarial parasite Plasmodium falciparum

The mechanism whereby heterozygous carriers of the sickle cell gene are protected against fatal malarial infections due to Plasmodium falciparum has been examined in a short term in vitro cultivation system. The results have show that both parasite invasion of red cells and parasite growth within red cells containing sickle haemoglobin (Hb-S) is restricted, but only under conditions of low (5%) oxygen tensions. To bring this about, the cells containing Hb-S need not sickle. Furthermore the growth retardation observed in the presence of Hb-S was also found to apply to the mature forms of the parasite. These findings offer a plausible mechanism for the protection of sickle heterozygotes against falciparum malaria.     

A procedure for the harvesting of mammalian plasmodia

Immunochemical research into the antigenic structure of a given disease agent presupposes the availability of undegraded antigen. Some types of immunochemical studies of plasmodia can be carried out with the intracellular parasites in situ in the host cell (for example, studies using the fluorescent antibody technique). In other techniques (such as double diffusion in gel, disc electrophoresis) the presence of host cell contaminants is undesirable, and these require to be reduced to a minimum.     

A compact semi-automated continuous cultivation system for Plasmodium falciparum

A compact inexpensive semi-automated continuous cultivation system for Plasmodium falciparum is described. The system is simple to operate and allows cultures to proceed unattended for several days. A particularly useful feature is a portable cultivation module that is freely transferable from the incubator to a tissue culture cabinet for manual operations such as sampling or addition of red cells. Each cultivation module holds plastic flasks which can give a combined culture surface area of up to 500 cm². Parasitaemias of 12% are attained routinely at red cell densities of 1 to 7 × 10⁸ per ml, if the medium is changed daily.     

Recent advances in malaria parasite cultivation and their application to studies on host-parasite relationships: a review

The continuous cultivation of the erythrocytic stages of Plasmodium falciparum was achieved in 1976 and techniques have now also been developed for continuous cultivation of these stages from P. knowlesi, P. fragile, P. inui, P. cynomolgi and P. berghei. The requisite conditions for successful cultivation are described. Gametes of certain isolates of P. falciparum can also now be produced in vitro and these are infective to mosquitos, leading to normal development of the parasite.     

Fansidar resistant falciparum malaria acquired in South East Asia

A case of Plasmodium falciparum malaria resistant to Fansidar (sulphadoxine plus pyrimethamine) at a level corresponding to R III and resistant to chloroquine is reported. The infection was most certainly acquired in Malaysia, but diagnosed and treated in a non-malarious area.Normal resorption and elimination rates of the Fansidar components excludes cure failure due to abnormal drug fate in the host.P. falciparum parasites from the patient have been maintained in in vitro cultures.The patient was permanently cured with mefloquine.     

The problem of cultivation of Mycobacterium leprae. A review with criteria for evaluating recent experimental work

Some criteria are presented to help evaluate papers appearing in the literature claiming successful cultivation of M. leprae either in the absence or in the presence of tissue-cultured cells. Recently, electron microscopic studies have definitely shown M. leprae to belong to the genus Mycobacterium and its division to occur through transverse section. A survey is given of the mycobacterial strains isolated in the last 10 years from leprosy lesions. These strains belong to taxonomically different species and cannot be considered to be M. leprae. No substantiated claim was made concerning the in vitro growth of M. leprae and the application of the tissue culture technique has been equally disappointing. The view is expressed that progress towards the in vitro cultivation of M. leprae can be made only as a result of increased knowledge about the intracellular environment and the metabolic activities of this organism, to be obtained by the application of modern biochemical and histochemical techniques.     

Comparative morphometric analysis of bloodstream and lymph forms of Trypanosoma (T.) brucei brucei grown in vitro and in vivo

The fine structure of bloodstream forms of Trypanosoma brucei cultivated in vitro, and of trypanosomes from lymph and blood of mammalian hosts, was compared morphometrically. The cell volume, quantitative parameters of the mitochondrion and of glycosomes were mainly investigated. A Coulter Channelyzer was used for the first time to measure the mean cell volume of living parasites. In vitro, the monomorphic trypanosomes between the feeder layer cells showed lower values for mitochondrial parameters than the slightly pleomorphic forms from the supernatant medium. Trypanosomes in culture were very similar morphologically to forms from lymph nodes of rats. Despite some morphometric differences between cultivated blood stream forms and those grown in vivo, the similarity of both populations was clear. Both populations, however, differed significantly from stages found in the vector or from procyclic culture forms.     

Non-specific cell mediated immunity in patients infected with Schistosoma mansoni in Kenya

Parameters of in vitro cell-mediated immunity (CMI) have been measured in the local Kenyan population infected with Schistosoma mansoni. Lymphocyte responses to the non-specific T cell mitogens Concanavalin A (Con A) and Phytohaemagglutinin (PHA) were reduced in about 60% of schistosomiasis patients. Lymphocytes from control uninfected, and S. mansoni-infected donors formed equal numbers of spontaneous rosettes with sheep red blood cells, indicating that there was no over-all reduction in the percentage numbers of T cells in the schistosomiasis patients.     

The primary isolation by haemoculture of Trypanosoma (Schizotrypanum) cruzi from animals and from man.

The results of the primary isolation of Trypansoma cruzi by haemoculture using a standard method, were successful for all of 14 experimentally infected mice, all naturally infected Didelphis azarae (eight animals: nine isolations) and six acute patients, but only for seven of 16 chronic patients. Two further chronic patients yielded positive cultures when the supernatant of slowly centrifuged blood fractions were inoculated.The low proportion of successful isolations from the group of chronic patients (maximum 56%) is probably mainly due to the low parasitaemia in this group.Haemoculture from the chronic patients also differed markedly from those from the other hosts in the very slow growth-rate obtained. This could not be explained solely by the small number of parasites inoculated. Serial ten-fold dilutions of infected mouse blood, down to 10^?5 ml of microscopically negative blood, did not prolong the in vitro pre-detection period of T. cruzi beyond two to four weeks, but the pre-detection in cultures from the chronic patients extended up to six months (range: six to 22 weeks, average 12 weeks).Factors that may be responsible for this phenomenon include unfavourable conditions in the culture media, the production of non-motile extra-cellular amastigotes (easily missed microscopically), the morphological characteristics of the original blood-stream trypomastigotes and humoral and cellular factors in the blood inoculated. Although some or all of these factors may contribute to the final outcome of haemoculture, consideration of the culture patterns observed suggests that the main factors responsible for the slow growth of T. cruzi in the cultures from chronic patients were the continuing activities of the humoral and cellular components in the blood inoculum.     

Comparison of whole blood cultures and peripheral blood mononuclear cell cultures for evaluation of lymphocyte reactivity during chronic schistosomiasis

Lymphocyte blastogenesis during chronic schistosomiasis has been evaluated previously by either whole blood or peripheral blood mononuclear cell (PBMN) culture techniques. These two methods were compared in the present study. Blastogenesis to the mitogen phytohaemagglutinin was greater in the whole blood cultures at the time assayed. Substantial antigen-induced lymphocyte transformation was elicited in both culture systems using cells from uninfected patients. However, patients' responses to schistosome-derived antigenic preparations as determined by whole blood culture were greatly reduced compared with PBMN cultures. In view of the known immunoregulatory activities associated with sera and cells from infected patients the whole blood technique may better reflect in vivo lymphocyte reactivity. The PBMN cultures are better suited to examine the immunoregulatory activities which may develop during this chronic infection.     

Chloroquine sensitivity of Plasmodium falciparum in Ibadan,Nigeria: II. Correlation of in vitrowith in vivo sensitivity

Plasmodium falciparum malaria was treated in 82 children with 25 mg/kg chloroquine orally over three days. They were observed for 28 days during which blood films were examined periodically for malaria parasites. Asexual forms of P. falciparum, present in the blood films of all the patients before commencing treatment, disappeared rapidly and by the third day no parasites were seen in blood films from any of them. Among the patients observed for more than three days, blood films remained negative throughout the observation period. In vitro tests of sensitivity of blood samples from 10 patients showed chloroquine concentrations of 0·5 to 0·8 nmol/ml to inhibit completely maturation from ring forms to schizonts.This suggests that P. falciparum in the Ibadan area is probably still fully sensitive to chloroquine.