Maturation of Plasmodium falciparum in multiply infected erythrocytes and the potential role in malaria pathogenesis.

Erythrocytes containing two or more parasites, referred to here as multiply infected erythrocytes (MIEs), are common in the blood of humans infected by Plasmodium falciparum. It is necessary to study these cells closely because the excess numbers of parasites they contain suggest that they could be overloaded with virulence factors. Here, microscopic examinations of blood smears from patients showed that up to seven merozoites can successfully invade an erythrocyte and mature to ring stage. However, in vitro culture showed that only up to three parasites can mature to late schizont stage. These observations were made by culturing the parasites in erythrocytes containing hemoglobin AA (HbAA), HbAS, and HbSS. Biochemical analysis of saponin-concentrated culture suggests that more hemozoin is produced in a MIE than in a singly infected erythrocyte (SIE). Studies have shown that ingestion of excessive hemozoin destroys monocytes and neutrophils, which could impair the immune system. Cultured parasites were also examined by transmission electron microscopy, and it was found that the quantity of knobs was dramatically increased on the membranes of erythrocytes containing multiple schizonts, compared to those containing only one schizont. Knobs contain, among other things, P. falciparum erythrocyte membrane protein 1 (PfEMP1) complex which mediates sequestration and promotes severe malaria. These findings suggest that P. falciparum increases its virulence by producing MIEs. On sexual life cycle of the parasite, microphotographs are presented in this report showing, for the first time, that two gametocytes can develop in one erythrocyte; they are referred to here as twin gametocytes. It is not known whether they can infect mosquitoes.

Possible relationship between Plasmodium falciparum ring-infected erythrocyte surface antigen (RESA) and host cell resistance to destruction by chemicals.

Repeated incubation of Plasmodium falciparum culture in 0.015% saponin solution for a total of 35 min destroys most of the uninfected cells, leaving only the ring-infected erythrocytes (RIEs). Parasites concentrated by this method can subsequently complete the asexual cycle and infect other erythrocytes. It is possible that resistance to saponin is mediated by one or more of the numerous parasite proteins present in the host erythrocyte membrane. We have found that schizonts are as susceptible as uninfected erythrocytes to saponin, indicating that the protective protein is parasite stage specific. Studies with cultured parasites have shown that ring-infected erythrocyte surface antigen (RESA) strengthens host erythrocyte membrane and protects against destruction. Therefore, we hypothesize that RESA could be involved in resistance to saponin. Here, we have carried out PCR test on RESA gene, using three different primers. One of them showed that P. falciparum isolates collected directly from infected humans and cultured only for a few days, or not at all, have amplicon sizes ranging from 372 to 510 bp. However, the amplicon size changed to 873 bp when in vitro growth was continued for one or more weeks. This genetic transformation precedes acquisition of the ability to confer saponin resistance to RIEs.

Hemozoin accumulation in Garnham bodies of Plasmodium falciparum gametocytes.

Garnham bodies are curious objects exclusive in erythrocytes containing sexual forms (gametocytes) of Plasmodium falciparum. Although the name is familiar, only a few photographs of Garnham bodies (G-bodies) have been published. Considering that other objects in malaria-infected erythrocytes, such as Schuffner's dots of Plasmodium vivax and Maurer's clefts of P. falciparum, have been found to have some functions, it has become necessary to pay closer attention to G-bodies. The present study presents previously unknown features of G-bodies and suggests a protective role for them. Wild isolates of P. falciparum were encouraged to grow in vitro under conditions that promote gametocytogenesis. Thin and thick smears of the cells were stained with Giemsa stain and examined under a light microscope. Production of G-bodies was detected in two isolates both in immature and mature gametocytes. Sometimes, the objects are found both at the top and below the parasite, contrary to previous suggestion of it being only on one side. They are highly diverse in morphology, including those that are shaped like m or S. Hemozoin accumulation was detected in some of the bodies, indicating direct opening into the cystoplasm of the parasite. It is possible that hemozoin was first produced in the parasite's food vacuole before being transported to G-bodies. Alternatively, hemoglobin transport vesicles could first accumulate in G-bodies where metabolically released ferriprotoporphyrin IX (FP) could be polymerized; but this would need acidic environment comparable to that in food vacuole. Electron microscopy has revealed that G-bodies consist of membranous whorls and it has been demonstrated experimentally that both infected and uninfected membranes promote ß-hematin formation. Whatever the mechanism, storing hemozoin in G-bodies outside the cytoplasm of the parasite could provide intraerythrocytic sexual forms of P. falciparum additional protection against FP toxicity.

Erythrocyte membranes convert monomeric ferriprotoporphyrin IX to ß-hematin in acidic environment at malarial fever temperature.

Hemozoin production makes it possible for intraerythrocytic malaria parasites to digest massive quantities of hemoglobin but still avoid potential ferriprotoporphyrin IX (FP) toxicity, which they cannot decompose further. Some antimalarial drugs, such as chloroquine, work by inhibiting this production, forcing the parasite to starve to death. As part of the efforts to identify possible biological mechanisms of FP polymerization, we have used normal human erythrocyte membranes as a model, to promote ß-hematin (ß-h) synthesis. Hemin in 35% aqueous dimethyl sulfoxide (DMSO) was reacted with isolated erythrocyte membranes and incubated overnight in sodium acetate buffer, pH 4.8, at 41°C. Infrared spectroscopy and electron microscopy showed that ß-h was produced. Hemin in 10% was less effective as the substrate than when it was in 35% DMSO. A high malarial temperature seemed to be necessary, because FP polymerization was less at 37°C than at 41°C. Production was partially inhibited by chloroquine. These observations are of interest because other investigators have reported that membrane lipids mediated FP polymerization, but whole membranes were ineffective. On the other hand, our hypothesis is that the transport vesicles (TV) in malaria parasites could provide the receptor for FP and the lipids that promote hemozoin formation. Erythrocyte membranes may not be directly involved, but Plasmodium species transport hemoglobin in membrane-bound TV into food vacuoles, where hemoglobin catabolism is completed and hemozoin crystals are stored.

Microscopic detection of mixed malarial infections: improvement by saponin hemolysis.

OBJECTIVE: The objective of the present study was to determine whether saponin hemolysis could improve microscopic detection of malaria parasites in human blood, since it has been previously reported that the technique has been used to enrich Plasmodium falciparum culture to >or=90% parasitemia. MATERIAL AND METHODS: Blood samples from suspected malaria cases were first examined in routine thick and thin smears under the microscope. The sample (1 ml) was then hemolyzed with 0.015% saponin in saline and centrifuged, the separated pellet was stained with Giemsa stain and examined microscopically, using PCR to confirm species identification. RESULTS: With P. falciparum in vitro culture, the proportions of infected erythrocytes were approximately 0.7-2% before and 65-97% after saponin hemolysis, confirming published reports. In contrast, there was little or no increase in the proportions of intact infected erythrocytes after saponin hemolysis of clinical blood specimens. However, 20-600 hemolyzed parasites were detected per field under the microscope after saponin hemolysis of patients' blood samples that contained only 1-15 parasites per field in conventional thick smears. In addition, more P. falciparum gametocytes were detected after saponin hemolysis. CONCLUSION: Saponin hemolysis concentrated the parasites in large volumes of blood into a small volume that could be smeared on a slide. This concentration method made it easy to detect malaria parasites and reduced the time needed for microscopy. In the present study, the method was comparable to PCR for the identification of P. vivax and P. falciparum mixed infections.

Requirements for maximal enrichment of viable intraerythrocytic Plasmodium falciparum rings by saponin hemolysis.

The purpose of the present study was to confirm the effectiveness of saponin hemolysis for concentrating ring-infected erythrocytes in Plasmodium falciparum cultures and to determine the actual numbers of the enriched parasites, not just percentage parasitemia. This is important because various molecular biology and vaccine development against malaria require useable quantities of pure culture with minimal number of uninfected erythrocytes at all stages. Synchronized cultures of three P. falciparum strains were exposed to 0.015% isotonic saponin solution for 30 minutes on ice. They were centrifuged and the pellets were treated again with saponin solution for 3-7 minutes. Initially, most of the cultures contained approximately 10(10) erythrocytes and 1-7% parasitemia, but at the end of the enrichment up to 10(8) of erythrocytes containing 90-99.8% parasitemia were recovered (maximal enrichment). From microscopic examination of the cells it was calculated that the hemolysis rate of uninfected and infected erythrocytes was circa 27 to 1, which could account for the enrichment. Studies by other investigators have suggested that P. falciparum merozoite invasion decreases erythrocyte membrane lipids, and it has been reported that reduction of membrane cholesterol could make erythrocytes saponin-resistant. The possibility that merozoite invasion made erythrocytes partially resistant to saponin hemolysis was strengthened by the observation that the proportions of multiple infections increased significantly in the enriched cultures. However, mature asexual parasites could not be concentrated by this method, suggesting possible differences between the membranes of erythrocytes containing ring forms and those of trophozoites and schizonts. Ring-infected erythrocytes freshly from malaria patients could also not be concentrated by the method described here, suggesting that the ability to induce saponin resistance in erythrocytes was acquired by the parasites in vitro.

Comparison of Plasmodium falciparum growth in sickle cells in low oxygen environment and candle-jar.

The first successful in vitro cultivation of Plasmodium falciparum in sickle cells in a gas mixture containing 3% oxygen, 4% carbon dioxide and 93% nitrogen has been reported recently, contradicting earlier claims that the parasite does not multiply continuously in sickle cell trait (HbAS) and sickle cell anemia (HbSS) erythrocytes at low oxygen tension. The present study extends that report by growing three P. falciparum strains in erythrocytes from four different sickle cell trait and four sickle cell anemia donors. Because P. falciparum is known to grow normally in sickle cells when incubated in a candle-jar estimated to contain 15-18% oxygen, we have also compared the growth at 3% oxygen with that in a candle-jar. For convenience, we also refer to the 3% oxygen and the candle-jar as low and high oxygen environment, respectively. The three P. falciparum strains were first grown continuously in low oxygen environment for at least 1 month in erythrocytes from one HbAS carrier. These stock cultures were then used to infect erythrocytes from additional three HbAS carriers and four HbSS patients. Results of the experiments showed that parasite growth and hemozoin production in HbAS erythrocytes in low oxygen environment were comparable to those obtained in the candle-jar. There was growth retardation in HbSS erythrocytes in low oxygen environment, but some of the parasites survived and eventually produced high parasitemia levels. Continuous cultivation of different P. falciparum strains in HbAS erythrocytes is necessary for investigation of possible molecular differences between malaria parasites in sickle cells and those in HbAA erythrocytes.