Low infectivity of Plasmodium falciparum gametocytes to Anopheles gambiae following treatment with sulfadoxine-pyrimethamine in Mali

Sulfadoxine-pyrimethamine (SP) treatment increases the rate of gametocyte carriage and selects SP resistance-conferring mutations in Plasmodium falciparum dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS), raising concerns of increased malaria transmission and spread of drug resistance. In a setting in Mali where SP was highly efficacious, we measured the prevalence of DHFR and DHPS mutations in P. falciparum infections with microscopy-detected gametocytes following SP treatment, and used direct feeding to assess infectivity to Anopheles gambiae sensu lato. Children and young adults presenting with uncomplicated malaria were treated with SP or chloroquine and followed for 28 days. Gametocyte carriage peaked at 67% 1 week after treatment with a single dose of SP. Those post-SP gametocytes carried significantly more DHFR and DHPS mutations than pre-treatment asexual parasites from the same population. Only 0.5% of 1728 mosquitoes fed on SP-treated gametocyte carriers developed oocysts, while 11% of 198 mosquitoes fed on chloroquine-treated gametocyte carriers were positive for oocysts. This study shows that in an area of high SP efficacy, although SP treatment sharply increased gametocyte carriage, the infectiousness of these gametocytes to the vector may be very low. Accurate and robust methods for measuring infectivity are needed to guide malaria control interventions that affect transmission.     

Plasmodium falciparum: mRNA co-expression and protein co-localisation of two gene products upregulated in early gametocytes

Genes encoding Plasmodium falciparum proteins Pfs16 and Pfpeg3/mdv1, specifically appearing in the parasitophorous vacuole of the early gametocytes, are upregulated at the onset of sexual differentiation. Analysis of asexual development in gametocyte producing and non-producing clones of P. falciparum indicated that these genes are also transcribed at a low level in asexual parasites, although their protein products are not detectable in these stages by immunofluorescence. Immunoelectron microscopic analysis of stage II gametocytes indicated that Pfs16 and Pfpeg3/mdv1 proteins co-localise in the parasitophorous vacuole membrane and in all derived membranous structures (such as the multi-laminate membrane whorls of the circular clefts in the infected erythrocyte cytoplasm and the membranes of the gametocyte food vacuoles). In this analysis both proteins were also observed for the first time in the membrane and in the lumen of distinct cleft-like structures in the erythrocyte cytoplasm.     

A study on pathogenicity and mosquito transmission success in the rodent malaria parasite Plasmodium chabaudi adami

We investigated the parasitology, pathogenicity (virulence) and infectivity to mosquitoes of blood infections in mice, of two strains, DS and DK, of the rodent malaria parasite Plasmodium chabaudi adami. Blood infections of DS were found to be highly pathogenic; the asexual parasites in these infections were fast-growing and showed no evidence of selectivity in their infection of host erythrocytes. In contrast to DS, blood infections of DK were much less pathogenic; the asexual parasites were slower-growing and showed a moderate degree of selectivity to a subset of erythrocytes which were not reticulocytes. In both DS and DK infections, infectivity to mosquitoes was highest before the peak of asexual parasitaemia had occurred; usually this did not coincide with the time when gametocyte numbers in the blood were highest. Infections with the pathogenic DS strain in CBA mice produced fewer gametocytes than did the less pathogenic DK strain. The DS strain infections in both CBA and C57 mice were also significantly much less infective to mosquitoes than the DK strain. Investigations by others on the related rodent malaria parasite subspecies, Plasmodium chabaudi chabaudi, have indicated that the mosquito infectivity of blood infections in mice tended to be higher in the more pathogenic (virulent) and lower in the less pathogenic strains of this parasite subspecies. This is the converse of the finding of the present investigation of blood infections of P. c. adami in mice in which a more pathogenic, or virulent, strain (DS) of these parasites was significantly much less infective to mosquitoes than was a less pathogenic strain (DK).     

The effect of chloroquine treatment on the infectivity of Plasmodium chabaudi gametocytes.

The antimalarial drug chloroquine has been reported to increase the infectivity of the forms of blood-stage malaria parasites (gametocytes) that are capable of infecting mosquito vectors. This effect has been demonstrated convincingly in the short term (12 h post treatment), although several authors have suggested infectivity enhancement a week or more after treatment. We carried out experiments to investigate the effects of chloroquine on the longer-term infectivity of gametocytes of the rodent malaria parasite, Plasmodium chabaudi, to Anopheles stephensi mosquitoes. Gametocytes of chloroquine-treated infections were significantly more infectious than untreated infections 6 and 7 days post-treatment, although not on days 8 and 9. However, this effect was most likely the result of a reduction in infectivity in untreated infections, caused by immune activity which was not so pronounced in chloroquine-treated infections. Gametocytaemia (gametocytes per r.b.c.) showed a strong positive and linear relationship with infectivity. Infectivity was not influenced by either asexual parasitaemia, asexual density or anaemia. Parsimonious interpretations of the effect of chloroquine on gametocyte infectivity are discussed.     

An integrated model of Plasmodium falciparum dynamics

The within-host and between-host dynamics of malaria are linked in myriad ways, but most obviously by gametocytes, the parasite blood forms transmissible from human to mosquito. Gametocyte dynamics depend on those of non-transmissible blood forms, which stimulate immune responses, impeding transmission as well as within-host parasite densities. These dynamics can, in turn, influence antigenic diversity and recombination between genetically distinct parasites. Here, we embed a differential-equation model of parasite-immune system interactions within each of the individual humans represented in a discrete-event model of Plasmodium falciparum transmission, and examine the effects of human population turnover, parasite antigenic diversity, recombination, and gametocyte production on the dynamics of malaria. Our results indicate that the local persistence of P. falciparum increases with turnover in the human population and antigenic diversity in the parasite, particularly in combination, and that antigenic diversity arising from meiotic recombination in the parasite has complex differential effects on the persistence of founder and progeny genotypes. We also find that reductions in the duration of individual human infectivity to mosquitoes, even if universal, produce population-level effects only if near-absolute, and that, in competition, the persistence and prevalence of parasite genotypes with gametocyte production concordant with data exceed those of genotypes with higher gametocyte production. This new, integrated approach provides a framework for investigating relationships between pathogen dynamics within an individual host and pathogen dynamics within interacting host and vector populations.     

Differential adhesive properties of sequestered asexual and sexual stages of Plasmodium falciparum on human endothelial cells are tissue independent

The protozoan parasite Plasmodium falciparum, responsible for the most severe form of malaria, is able to sequester from peripheral circulation during infection. The asexual stage parasites sequester by binding to endothelial cell receptors in the microvasculature of various organs. P. falciparum gametocytes, the developmental stages responsible for parasite transmission from humans to Anopheles mosquitoes, also spend the almost ten days necessary for their maturation sequestered away from the peripheral circulation before they are released in blood mainstream. In contrast to those of asexual parasites, the mechanisms and cellular interactions responsible for immature gametocyte sequestration are largely unexplored, and controversial evidence has been produced so far on this matter. Here we present a systematic comparison of cell binding properties of asexual stages and immature and mature gametocytes from the reference P. falciparum clone 3D7 and from a patient parasite isolate on a panel of human endothelial cells from different tissues. This analysis includes assays on human bone marrow derived endothelial cell lines (HBMEC), as this tissue has been proposed as a major site of gametocyte maturation. Our results clearly demonstrate that cell adhesion of asexual stage parasites is consistently more efficient than that, virtually undetectable of immature gametocytes, irrespectively of the endothelial cell lines used and of parasite genotypes. Importantly, immature gametocytes of both lines tested here do not show a higher binding efficiency compared to asexual stages on bone marrow derived endothelial cells, unlike previously reported in the only study on this issue. This indicates that gametocyte-host interactions in this tissue are unlikely to be mediated by the same adhesion processes to specific endothelial receptors as seen with asexual forms.     

Plasmodium falciparum gametocytes: their longevity and infectivity.

The longevity and infectivity of isolated populations of Plasmodium falciparum gametocytes were studied. Following chloroquine treatment gametocyte numbers fell with a constant rate of loss over a period of 16-24 days; the populations had a half-life of 2-4 days. The sex ratio stayed constant throughout at 4 female: 1 male. The ability of the microgametocytes to exflagellate and the infectivity of the population to mosquitoes persisted for 3 weeks. Antibodies to the gametocytes were detected but not in every patient studied. It was concluded that the gametocytes of P. falciparum are both long-lived and show persistent infectivity to mosquitoes. They can stimulate antibody production but the immune response appears to play no part in their elimination, which probably takes place in the spleen as a part of the normal process of removing old, damaged and malformed red cells.     

Human immune responses that reduce the transmission of Plasmodium falciparum in African populations

Malaria-infected individuals can develop antibodies which reduce the infectiousness of Plasmodium gametocytes to biting Anopheles mosquitoes. When ingested in a bloodmeal together with gametocytes, these antibodies reduce or prevent subsequent parasite maturation in the insect host. This transmission-blocking immunity is usually measured in human sera by testing its effect on the infectivity of gametocytes grown in vitro. Here we evaluate evidence of transmission-blocking immunity in eight studies conducted in three African countries. Plasmodium falciparum gametocytes isolated from each individual were fed to mosquitoes in both autologous plasma collected with the parasites, and permissive serum from non-exposed donors. Evidence of transmission reducing effects of autologous plasma was found in all countries. Experiments involving 116 Gambian children (aged 0.5-15 years) were combined to determine which factors were associated with transmission reducing immune responses. The chances of infecting at least one mosquito and the average proportion of infected mosquitoes were negatively associated with recent exposure to gametocytes and sampling late in the season. These results suggest that effective malaria transmission-reducing antibodies do not commonly circulate in African children, and that recent gametocyte carriage is required to initiate and/or boost such responses.     

The epidemiology of Plasmodium falciparum gametocytes: weapons of mass dispersion

Much of the epidemiology of Plasmodium falciparum in Sub-Saharan Africa focuses on the prevalence patterns of asexual parasites in people of different ages, whereas the gametocytes that propagate the disease are often neglected. One expected benefit of the widespread introduction of artemisinin-based combination therapy for malaria is a reduction in gametocyte carriage. However, the factors that affect the transmission of parasites from humans to mosquitoes show complex dynamics in relation to the intensity and seasonality of malaria transmission, and thus such benefits might not be automatic. Here, we review data on gametocyte carriage in the context of the development of naturally acquired immunity and population infectivity.     

A developmental defect in Plasmodium falciparum male gametogenesis

Asexually replicating populations of Plasmodium parasites, including those from cloned lines, generate both male and female gametes to complete the malaria life cycle through the mosquito. The generation of these sexual forms begins with the induction of gametocytes from haploid asexual stage parasites in the blood of the vertebrate host. The molecular processes that govern the differentiation and development of the sexual forms are largely unknown. Here we describe a defect that affects the development of competent male gametocytes from a mutant clone of P. falciparum (Dd2). Comparison of the Dd2 clone to the predecessor clone from which it was derived (W2'82) shows that the defect is a mutation that arose during the long-term cultivation of asexual stages in vitro. Light and electron microscopic images, and indirect immunofluorescence assays with male-specific anti-alpha- tubulin II antibodies, indicate a global disruption of male development at the gametocyte level with at least a 70-90% reduction in the proportion of mature male gametocytes by the Dd2 clone relative to W2'82. A high prevalence of abnormal gametocyte forms, frequently containing multiple and unusually large vacuoles, is associated with the defect. The reduced production of mature male gametocytes may reflect a problem in processes that commit a gametocyte to male development or a progressive attrition of viable male gametocytes during maturation. The defect is genetically linked to an almost complete absence of male gamete production and of infectivity to mosquitoes. This is the first sex-specific developmental mutation identified and characterized in Plasmodium.     

The effect of partial host immunity on the transmission of malaria parasites.

Experiments were carried out to determine the effect of partial host immunity against the rodent malaria parasite Plasmodium chabaudi on the transmission success of the parasite. There was a fourfold reduction in both the blood-stage, asexually replicating parasite density and the gametocyte (transmissable stage) density in immunized hosts. Some of the reduction in asexual parasite densities was due to strain-specific immunity, but there was no evidence that strain-specific immunity affected gametocyte densities. However, immunity did affect transmission in a strain-specific manner, with a fivefold reduction in gametocyte infectivity to mosquitoes in homologous challenges compared with heterologous challenges or non-immunized controls. This implies the existence of a mechanism of strain-specific infectivity-reducing immunity that does not affect the density of gametocytes circulating in peripheral blood. The proportion of asexual parasites that produced gametocytes increased during the course of infection in both non-immunized and in immunized hosts, but immunity increased gametocyte production early in the infection.     

Decreased hemoglobin concentrations,hyperparasitemia, and severe malaria are associated with increased Plasmodium falciparum gametocyte carriage

To determine factors influencing gametocyte carriage, a cross-sectional study was conducted among 512 patients admitted for Plasmodium falciparum malaria. After adjustments for potential confounders, hemoglobin concentrations were lower in gametocyte carriers 10.5 (+/-2.5) than in patients without gametocytes 12.5 (+/-2.3) (P < 0.0001). Hemoglobin concentrations were negatively correlated with peak gametocyte counts (Spearman's p = -0.37, P < 0.0001) and gametocyte carriage durations (Spearman's p = -(0.30, P < 0.0001). Adjustments for the duration of the malaria episode and other potential confounders did not alter the association (P < 0.0001). After adjustment for potential confounders, the median asexual parasitemia was higher in patients with gametocytes than in patients without gametocytes (P = 0.003). Severe malaria cases were more likely to have gametocytes (65%) than malaria with hyperparasitemia (38%) or mild malaria (31%) (P = 0.0001). These findings suggest that events surrounding anemia and tissue hypoxia stimulate Plasmodium falciparum gametocytogenesis.     

Appropriate Time for Primaquine Treatment to Reduce Plasmodium falciparum Transmission in Hypoendemic Areas

Artemesinin-combination therapies (ACT) for falciparum malaria reduce gametocyte carriage, and therefore reduce transmission. Artemisinin derivatives will act against only young gametocytes whereas primaquine acts on mature gametocytes which are present usually in the circulation at the time when the patient presents for treatment. Both artemisinin derivatives and primaquine have short half-lives, less than 1 hr and 7 hr, respectively. Therefore, asexual parasites or young gametocytes remain after completed ACT. A single dose of primaquine (0.50-0.75 mg base/kg) at the end of ACT can kill only mature gametocytes but cannot kill young gametocytes (if present). Remaining asexual forms after completion of ACT course, e.g., artesunate-mefloquine for 3 days, may develop to mature gametocytes 7-15 days later. Thus, an additional dose of primaquine (0.50-0.75 mg base/kg) given 2 weeks after ACT completion may be beneficial for killing remaining mature gametocytes and contribute to more interruption of Plasmodium falciparum transmission than giving only 1 single dose of primaquine just after completing ACT.     

The course of infections and pathology in immunomodulated NOD/LtSz-SCID mice inoculated with Plasmodium falciparum laboratory lines and clinical isolates

Human chimeras are potentially invaluable models for hemoprotozoan parasites such as Plasmodium falciparum. The work presented assesses the susceptibility of immunomodulated NOD/LtSz-SCID mice to genetically distinct P. falciparum parasites. To this end, mice grafted with human erythrocytes were inoculated with two P. falciparum laboratory lines, 3D7 and Dd2 and four clinical isolates, ISCIII-230, ISCIII-231, ISCIII-381 and ISCIII-399. The results showed that, without a previous period of parasite adaptation, 100% of the inoculated mice developed an infection, generally self-limited, though some mice died. The parasitemias ranged from 0.05 to 8% and lasted an average of 19 days (15-26 days) depending on the line or isolate studied. Sexual forms of different maturity, stage II-IV and mature gametocytes were observed in the peripheral blood of mice in 22, 50, 25, 72 and 80% of the mice infected with Dd2, ISCIII-399, ISCIII-230, ISCIII-231 and ISCIII-381 isolates, respectively. The study of the clinical symptoms, the haematological parameters and the histopathological changes in the infected mice showed that most of the malaria features were present in the infected mice except that the sequestration of infected erythrocytes was absent or at most a minor phenomenon, as also indicated by the presence of mature forms of the parasites in the peripheral blood. This study shows that the human chimeras allow the complete asexual and sexual erythrocytic cycle of different P. falciparum lines and clinical isolates to be observed in vivo. It opens a new way to investigate any parasite population in terms of infectivity, transmission, and drug resistance.     

Blockade of TNF receptor 1 reduces disease severity but increases parasite transmission during Plasmodium chabaudi chabaudi infection

Reducing host carriage of transmission-stage malaria parasites (gametocytes) is expected to decrease the population-wide burden of malaria. Some malaria disease severity is attributed to the induction of the pro-inflammatory cytokines TNF-alpha and lymphotoxin-alpha (LT-alpha), and we are interested in whether anti-malaria interventions which ameliorate the symptoms induced by those cytokines may have the capacity to alter malaria transmission. As many functions of TNF-alpha and LT-alpha are exerted through TNF receptor 1 (TNFR1), we investigated the effect TNFR1 blockade exerted on parasite transmission using the rodent malaria Plasmodium chabaudi chabaudi. We found that blocking TNFR1 simultaneously increased gametocyte density and infectivity to mosquitoes, whilst reducing disease severity (weight loss). These transmission-enhancing and severity-reducing effects of TNFR1 blockade were independent of asexual parasite load and were observed for several P. c. chabaudi genotypes. These results suggest that the effects of candidate malaria interventions on infectivity should be examined alongside effects on disease severity so that the epidemiological consequences of such interventions can be evaluated.