Short-term effect of chloroquine on the infectivity of Plasmodium chabaudi gametocytes.

The short-term enhancing effect of chloroquine on gametocyte infectivity was investigated with Plasmodium chabaudi chabaudi, a synchronous parasite which is highly sensitive to chloroquine. In comparison with control groups, oocyst numbers increased in mosquitoes fed on mice 12 hours after the injection of 5 mg/kg chloroquine (180% of controls) although it was not statistically significant. No effect was seen with 1 mg/kg chloroquine. The authors interpretation is that chloroquine impaired the schizogony, thus reducing also the release of toxic material of parasite origin which blocks gametocytes infectivity. Results of similar experiments with other rodent species of Plasmodium are compared and discussed in relation with the chronobiological characteristics of these parasites.     

Plasmodium cynomolgi: gametocytocidal activity of the anti-malarial compound CDRI 80/53 (elubaquine) in rhesus monkeys

The gametocytocidal action of a new enamine analogue of primaquine, elubaquine (compound CDRI 80/53, bulaquine), has been evaluated against Plasmodium cynomolgi B in rhesus monkeys. Colony bred Anopheles stephensi mosquitoes were fed on gametocyte carrying rhesus monkeys prior to and at varying intervals after oral administration of a single dose of elubaquine at doses ranging between 0.63 and 5.00 mg/kg. Complete loss of oocyst development and mosquito infectivity was observed within 24 h after administering a single 1.25 mg/kg dose, while higher dose of 3.75 mg/kg inhibited oocyst development within 5 h, indicating gametocytocidal action of the compound. Elubaquine did not show any action against developing oocysts in the vector.     

Plasmodium chabaudi: effect of antimalarial drugs on gametocytogenesis.

The proportion of asexual blood-stage malaria parasites that develop into transmission stages (gametocytes) can increase in response to stress. We investigated whether stress imposed by a variety of antimalarial drugs administered before or during infection increased gametocyte production (gametocytogenesis) in vivo in the rodent malaria parasite, Plasmodium chabaudi. All methods of drug treatment greatly reduced the numbers of asexual parasites produced during an infection but resulted in either no reduction in numbers of gametocytes or a smaller reduction than that experienced by asexuals. We used a simple model to estimate temporal variation in gametocyte production. Temporal patterns of gametocytogenesis did not greatly differ between untreated and prophylaxis infections, with rates of gametocytogenesis always increasing as the infection progressed. In contrast, administration of drugs 5 days after infection stimulated increased rates of gametocytogenesis early in the infection, resulting in earlier peak gametocyte densities relative to untreated infections. Given the correlation between gametocyte densities and infectivity to mosquito vectors, and the high frequency of subcurative drug therapy and prophylaxis in human populations, these data suggest that antimalarial drugs may frequently have only a small effect on reducing malaria transmission and may help to explain the rapid spread of drug-resistant geno-types.     

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.     

Plasmodium chabaudi: in vivo effects of Ca2+ antagonists on chloroquine-resistant and chloroquine-sensitive parasites

The effects of Ca2+ antagonists, verapamil, nicardipine, and diltiazem, on susceptibility to chloroquine were examined in mice infected with chloroquine-sensitive and chloroquine-resistant lines of Plasmodium chabaudi. In mice that received no chloroquine, daily injections of 50 mg/kg of verapamil, nicardipine, or diltiazem did not affect the growth of both sensitive and resistant parasites. When mice were injected daily with verapamil plus 2 to 3 mg/kg chloroquine, the chloroquine-sensitive parasite became more susceptible to chloroquine than the parasite in mice given chloroquine alone. On the other hand, in mice infected with chloroquine-resistant parasites, verapamil severely suppressed the growth of the parasite when accompanied by daily injections of 2 to 3 mg/kg of chloroquine, at which doses resistant parasites grew steadily in the absence of verapamil, indicating reversal of chloroquine resistance. This reversal was dose-dependent between 5 and 50 mg/kg of verapamil. Daily injections of nicardipine or diltiazem at 50 mg/kg also reversed resistance to chloroquine in resistant parasites. These results indicate that Ca2+ antagonists increase the susceptibility to chloroquine in a sensitive line of P. chabaudi and reverse chloroquine resistance in a resistant line.     

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.     

Adaptive changes in Plasmodium transmission strategies following chloroquine chemotherapy.

Both theory and data suggest that malaria parasites divert resources from within-host replication to the production of transmission stages (gametocytes) when conditions deteriorate. Increased investment into transmission stages should therefore follow subcurative treatment with antimalarial drugs, but relevant clinical studies necessarily lack adequate control groups. We therefore carried out controlled experiments to test this hypothesis, using a rodent malaria (Plasmodium chabaudi) model. Infections treated with a subcurative dose of the antimalarial chloroquine showed an earlier peak and a greater rate of gametocyte production relative to untreated controls. These alterations led to correlated changes in infectivity to mosquitoes, with the consequence that chloroquine treatment had no effect on the proportion of mosquitoes infected. Treatment of human malaria commonly does not result in complete parasite clearance. If surviving parasites produce compensatory increases in their rate of gametocyte production similar to those reported here, such treatment may have minimal effect on decreasing, and may actually increase, transmission. Importantly, if increased investment in transmission is a generalized stress response, the effect might be observed following a variety of antimalarial treatments, including other drugs and potential vaccines. Similar parasite life history counter-adaptations to intervention strategies are likely to occur in many disease-causing organisms.     

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.     

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).     

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.     

The infectivity of Plasmodium yoelii in different strains of mice

We evaluated the effect of using Medium 199 alone and Medium 199 supplemented with 5% normal mouse serum, 5% fetal calf serum, 5% bovine serum albumin or 5% Albumax on Plasmodium yoelii sporozoite yield from infected mosquitoes and infectivity in BALB/c mice. The sporozoites yield, as well as their infectivity, was statistically lower (P = 0.0031) when unsupplemented Medium 199 was used to separate sporozoites from infected mosquitoes. Although Medium 199 supplemented with Albumax led to lower sporozoite yield (P < 0.0009), infectivity of the sporozoites was similar to those obtained with the other medium supplements. Because normal mouse serum supports good sporozoite infections and is also the supplement that can be used repeatedly in mice during multiple sporozoite injections without inducing anaphylaxis, we selected it to evaluate the infectivity of P. yoelii sporozoites in different strains of mice. After injecting mice with serial dilutions of sporozoites and detecting patent infections, we determined that the infective dose 50 (ID50) for BALB/c, C57Bl/6, A/J, and B10BR mice ranged between 4.9 and 10.6 sporozoites. The ID50 obtained for CD-1 mice (147 sporozoites) was significantly higher.     

Sulfadoxine-pyrimethamine impairs Plasmodium falciparum gametocyte infectivity and Anopheles mosquito survival

Sulfadoxine-pyrimethamine (SP) is currently the drug of choice for intermittent preventive treatment of Plasmodiumfalciparum both in pregnancy and infancy. A prolonged parasite clearance time conferred by dhfr and dhps mutations is believed to be responsible for increased gametocyte prevalence in SP treated individuals. However, using a direct feeding assay in Mali, we showed that gametocytes present in peripheral venous blood post-SP treatment had reduced infectivity for Anophelesgambiae sensu stricto (ss) mosquitoes. We investigated the potential mechanisms involved in the dhfr and dhps quintuple mutant NF-135 and the single dhps 437 mutant NF-54. Concentrations of sulfadoxine (S) and pyrimethamine (P) equivalent to the serum levels of the respective drugs on day 3 (S = 61 μg/ml, P = 154.7 ng/ml) day 7 (S = 33.8 μg/ml, P = 66.6 ng/ml) and day 14 (S = 14.2 μg/ml, P = 15.7 ng/ml) post-SP treatment were used to study the effect on gametocytogenesis, gametocyte maturation and infectivity to Anophelesstephensi mosquitoes fed through an artificial membrane. The drugs readily induced gametocytogenesis in the mutant NF-135 strain but effectively killed the wild-type NF-54. However, both drugs impaired gametocyte maturation yielding odd-shaped non-exflagellating mature gametocytes. The concomitant ingestion of both S and P together with gametocytemic blood-meal significantly reduced the prevalence of oocyst positivity as well as oocyst density when compared to controls (P < 0.001). In addition, day 3 concentrations of SP decreased mosquito survival by up to 65% (P < 0.001). This study demonstrates that SP is deleterious in vitro for gametocyte infectivity as well as mosquito survival.     

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.     

Population dynamics of Plasmodium falciparum sporogony in laboratory-infected Anopheles gambiae.

The population dynamics of cultured Plasmodium falciparum parasites was examined during their sporogonic development in Anopheles gambiae mosquitoes. Estimates of absolute densities were determined for each life stage, and life tables were constructed for each of 38 experimental infections. Macrogametocyte and ookinete mortalities contributed equally to the overall mortality. On average, there was a 40-fold decrease in parasite numbers in the transition from the macrogametocyte to the ookinete stage, a 69-fold decrease in the transition from ookinete to oocyst stages, and a total net decrease in parasite numbers from macrogametocyte to oocyst stage of 2,754-fold (i.e., multiplicative). There was no relationship between macrogametocyte and ookinete densities due to the inherent variability in fertility among different gametocyte cultures. There was a curvilinear relationship (r2 = 0.66) between ookinete and oocyst densities. Above a threshold of about 30 ookinetes/mosquito, the oocyst yield per ookinete became increasingly greater with increasing ookinete density. There was a linear relationship (r2 = 0.73) between oocyst and sporozoite densities, with an average of 663 salivary gland sporozoites produced per oocyst. Sporozoite production per oocyst was not affected by oocyst density and virtually all oocyst infections resulted in sporozoite infections of the salivery glands. This quantitative study indicates that the sporogony of cultured P. falciparum in laboratory-infected A. gambiae is an inefficient process and that the ookinete is the key transitional stage affecting the probability of vector infectivity.     

Effects of chloroquine and sulfadoxine/pyrimethamine on gametocytes in patients with uncomplicated Plasmodium falciparum malaria in Colombia

The effect of antimalarials on gametocytes can influence transmission and the spread of drug resistance. In order to further understand this relationship, we determined the proportion of gametocyte carriers over time post-treatment in patients with uncomplicated Plasmodium falciparum malaria who were treated with either chloroquine (CQ) or sulfadoxine/pyrimethamine (SP). The overall proportion of gametocyte carriers was high (85%) and not statistically significantly different between the CQ and SP treatment groups. However, an increased risk of carrying gametocytes on day 14 of follow up (1.26 95% CI 1.10-1.45) was found among patients having therapeutic failure to CQ compared with patients having an adequate therapeutic response. This finding confirms and extends reports of increased risk of gametocytaemia among CQ resistant P. falciparum.     

Wolbachia strain wAlbB enhances infection by the rodent malaria parasite Plasmodium berghei in Anopheles gambiae mosquitoes

Wolbachia, a common bacterial endosymbiont of insects, has been shown to protect its hosts against a wide range of pathogens. However, not all strains exert a protective effect on their host. Here we assess the effects of two divergent Wolbachia strains, wAlbB from Aedes albopictus and wMelPop from Drosophila melanogaster, on the vector competence of Anopheles gambiae challenged with Plasmodium berghei. We show that the wAlbB strain significantly increases P. berghei oocyst levels in the mosquito midgut while wMelPop modestly suppresses oocyst levels. The wAlbB strain is avirulent to mosquitoes while wMelPop is moderately virulent to mosquitoes pre-blood meal and highly virulent after mosquitoes have fed on mice. These various effects on P. berghei levels suggest that Wolbachia strains differ in their interactions with the host and/or pathogen, and these differences could be used to dissect the molecular mechanisms that cause interference of pathogen development in mosquitoes.     

The transmission-blocking effects of antimalarial drugs revisited: fitness costs and sporontocidal effects of artesunate and sulfadoxine-pyrimethamine

Assays used to evaluate the transmission-blocking activity of antimalarial drugs are largely focused on their potential to inhibit or reduce the infectivity of gametocytes, the blood stages of the parasite that are responsible for the onward transmission to the mosquito vector. For this purpose, the drug is administered concomitantly with gametocyte-infected blood, and the results are evaluated as the percentage of reduction in the number of oocysts in the mosquito midgut. We report the results of a series of experiments that explore the transmission-blocking potential of two key antimalarial drugs, artesunate and sulfadoxine-pyrimethamine, when administered to mosquitoes already infected from a previous blood meal. For this purpose, uninfected mosquitoes and mosquitoes carrying a 6 day old Plasmodium relictum infection (early oocyst stages) were allowed to feed either on a drug-treated or an untreated host in a fully factorial experiment. This protocol allowed us to bypass the gametocyte stages and establish whether the drugs have a sporontocidal effect, i.e. whether they are able to arrest the ongoing development of oocysts and sporozoites, as would be the case when a mosquito takes a post-infection treated blood meal. In a separate experiment, we also explored whether a drug-treated blood meal impacted key life history traits of the mosquito relevant for transmission, and if this depended on their infection status. Our results showed that feeding on an artesunate- or sulfadoxine-pyrimethamine-treated hosts has no epidemiologically relevant effects on the fitness of infected or uninfected mosquitoes. In contrast, when infected mosquitoes fed on an sulfadoxine-pyrimethamine-treated host, we observed both a significant increase in the number of oocysts in the midgut, and a drastic decrease in both sporozoite prevalence (?30%) and burden (?80%) compared with the untreated controls. We discuss the potential mechanisms underlying these seemingly contradictory results and contend that, provided the results are translatable to human malaria, the potential epidemiological and evolutionary consequences of the current preventive use of sulfadoxine-pyrimethamine in malaria-endemic countries could be substantial.