Augustine U. Orjih

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 β-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 β-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 β-hematin (β-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 β-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.

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.

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 1010 erythrocytes and 1–7% parasitemia, but at the end of the enrichment up to 108 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.

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 ≧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.

Contents Vol. 17, 2008

507 Abstracts of Award-Winning Posters, 13th Annual Health Sciences Poster Conference, Faculty of Medicine, Health Sciences Centre, Kuwait University, Kuwait, April 22–24, 2008 512 Abstracts of Theses Approved for the MSc and PhD Degrees at the Faculty of Medicine, Health Sciences Centre, Kuwait University, Kuwait 515 List of Reviewers Vol. 17, 2008 517 Author Index Vol. 17, 2008 519 Subject Index Vol. 17, 2008