Jan Peter Verhave

The production of mature gametocytes of Plasmodium falciparum in continuous cultures of different isolates infective to mosquitoes

In vitro gametocytogenesis of Plasmodium falciparum was observed in all 22 isolates established in this laboratory. Gametocytes were produced in variable numbers--up to 3% of red cells--for a limited period of time after which this stage was seen only very sporadically. Complete maturation of microgametocytes in vitro was obtained in all 14 of the isolates that were tested for exflagellation. Up to 88.2% of membrane-fed Anopheles stephensi were infected from material produced in culture. It was also possible to infect A. gambiae and A. freeborni. Addition of fresh red cells and serum to culture material promoted infectivity of gametocytes. Gametocyte infectivity declined rapidly with time in the membrane feeders held at 38 degrees C.

Substantial Contribution of Submicroscopical Plasmodium falciparum Gametocyte Carriage to the Infectious Reservoir in an Area of Seasonal Transmission

Background Man to mosquito transmission of malaria depends on the presence of the sexual stage parasites, gametocytes, that often circulate at low densities. Gametocyte densities below the microscopical threshold of detection may be sufficient to infect mosquitoes but the importance of submicroscopical gametocyte carriage in different transmission settings is unknown. Methodology/Principal Findings Membrane feeding experiments were carried out on 80 children below 14 years of age at the end of the wet season in an area of seasonal malaria transmission in Burkina Faso. Gametocytes were quantified by microscopy and by Pfs25-based quantitative nucleic acid sequence-based amplification assay (QT-NASBA). The childrens infectiousness was determined by membrane feeding experiments in which a venous blood sample was offered to locally reared Anopheles mosquitoes. Gametocytes were detected in 30.0% (24/80) of the children by microscopy compared to 91.6% (65/71) by QT-NASBA (p<0.001). We observed a strong association between QT-NASBA gametocyte density and infection rates (p = 0.007). Children with microscopically detectable gametocytes were more likely to be infectious (68.2% compared to 31.7% of carriers of submicroscopical gametocytes, p = 0.001), and on average infected more mosquitoes (13.2% compared to 2.3%, p<0.001). However, because of the high prevalence of submicroscopical gametocyte carriage in the study population, carriers of sub-microscopical gametocytes were responsible for 24.2% of the malaria transmission in this population. Conclusions/Significance Submicroscopical gametocyte carriage is common in an area of seasonal transmission in Burkina Faso and contributes substantially to the human infectious reservoir. Submicroscopical gametocyte carriage should therefore be considered when implementing interventions that aim to reduce malaria transmission.

Effect of gametocyte sex ratio on infectivity of Plasmodium falciparum to Anopheles gambiae

Insectary-reared Anopheles gambiae were experimentally fed with the blood of 90 naturally infected human volunteers carrying gametocytes of Plasmodium falciparum. At least one mosquito was successfully infected in 74% of experiments. The probability that a gametocyte carrier was infective, the probability that a mosquito became infected, and the number of oocysts harboured were related to gametocyte density. The mean proportion of male gametocytes was 0.217 (i.e., 3.6 females for every male). Sex ratios differed significantly between gametocyte carriers. Variation in sex ratio was not related to the probability that a gametocyte carrier was infective. Among infective people whose sex ratio estimates were based on a reasonable number of gametocytes, sex ratio significantly predicted the proportion of infected mosquitoes and mean oocyst load, with infectivity rising as the proportion of the male gametocytes increased towards 50%. There was no indication that infectivity reached a peak at some intermediate sex ratio, as would be expected if random mating of gametes was the primary determinant of fertilization success. These results raise 2 interesting questions: why should higher sex ratios be more infective, and why is the observed population sex ratio lower than that which produces the greatest infectivity?

Malaria sporozoite penetration: a new approach by double staining

To determine, whether a sporozoite is outside the hepatocyte membrane or internalized, a double staining test was carried out using, successively, antibody labeled with peroxidase and fluorescein. This test permits the quantification of sporozoite entry and outline sporozoite-hepatocyte interactions.

The early sporogonic cycle of Plasmodium falciparum in laboratory‐infected Anopheles gambiae: an estimation of parasite efficacy

This study investigated the successive losses in the parasite densities of Plasmodium falciparum stages during the early sporogony in laboratory‐reared Anopheles gambiae infected by membrane feeding with blood from naturally infected gametocyte carriers (>50 gametocytes/mm3). The developmental stages of P. falciparum in the mosquito were studied from zygote to oocyst, by immunofluorescent method using monoclonal antibodies against the Pfs25 protein present on the surface of newly formed gametes. This method allows for assessment of the various sporogonic stages before, during and after passage of the midgut wall. Parasite densities were determined within the entire blood meal at 3 h (zygotes and macrogametes) and 24 h (ookinetes) post‐infection. At 48 h after the mosquito blood meal, midguts were checked for the presence of early oocysts. For the mid‐size oocysts count, classic microscopy examination was used at day 7 post‐infection. The parasite efficacy was estimated by following successive losses in parasite densities between different early stages of the sporogonic cycle in A. gambiae. Thirty‐seven experimental infections were realized with high gametocyte densities, ranging from 64 to 2392 gametocytes/mm3. All gametocyte carriers showed infection with round forms 100%; ookinetes were found in 91.9%. The prevalences of infections with oocysts were 48.6% at day 2 (young oocyst) and 37.8% at day 7 (mid‐size oocyst). The mean densities per mosquito for each parasite stage were 12.6 round forms, 5.5 ookinetes, 1.8 young oocyst and 2 mid‐size oocysts. Significant correlations were found between two consecutive parasite stages (round forms/ookinetes, ookinetes/young oocysts, young oocysts/mid‐size oocysts) and between round forms and mid‐size oocysts. The mean parasite density significantly decreased between round forms and ookinetes (yield Y1 = 41.6%) and between ookinetes and young oocysts (Y2 = 61.4%). By contrast, no significant decrease was observed between young oocysts and mid‐size oocysts (Y3 = 91.2%). The overall yield of the early sporogonic cycle (from round form to oocyst at day 7) was equal to 25.7%, indicating that almost 3/4 of the total parasites were lost during the early step of the sporogonic cycle, from 3 h post‐infection to day 7.

Exoerythrocytic Development of Malarial Parasites

Publisher Summary This chapter discusses the exoerythrocytic development of malarial parasites and summarizes the knowledge of the exo-erythrocytic (EE) form, a stage in the life-cycle of Plasmodium. In describing the development of the intracellular EE stage, it should be realized that, at the moment of fixation, the parasites are not resting stages but actively growing forms. Because of the importance of the EE cycle in the establishment of a blood infection, this chapter considers the EE form the original or primitive stage, a bridgehead by which Plasmodium captures the vertebrate host. This chapter also includes a historical survey, the morphology of the exo-erythrocytic liver stage, the metabolism of the exo-erythrocytic liver stage, and the cultivation of the exo-erythrocytic liver stage. The rodent EE forms, and their development from sporozoites in hepatocytes have been studied more extensively in recent years. Only very recently the primate EE parasites have been yielded to fine structural studies, allowing more knowledge to become available.

Dynamics of the Human Infectious Reservoir for Malaria Determined by Mosquito Feeding Assays and Ultrasensitive Malaria Diagnosis in Burkina Faso

BACKGROUND Plasmodium falciparum gametocytes are essential for malaria transmission. Malaria control measures that aim at reducing transmission require an accurate characterization of the human infectious reservoir. METHODS We longitudinally determined human infectiousness to mosquitoes and P. falciparum carriage by an ultrasensitive RNA-based diagnostics in 130 randomly selected inhabitants of an endemic area. RESULTS At least 1 mosquito was infected by 32.6% (100 of 307) of the blood samples; in total, 7.6% of mosquitoes (916 of 12 079) were infected. The proportion of infectious individuals and infected mosquitoes were negatively associated with age and positively with asexual parasites (P < .001). Human infectiousness was higher at the start of the wet season and subsequently declined at the peak of the wet season (adjusted odds ratio, 0.52; P = .06) and in the dry season (0.23; P < .001). Overall, microscopy-negative individuals were responsible for 28.7% of infectious individuals (25 of 87) and 17.0% of mosquito infections (145 of 855). CONCLUSIONS Our study reveals that the infectious reservoir peaks at the start of the wet season, with prominent roles for infections in children and submicroscopic infections. These findings have important consequences for strategies and the timing of interventions, which need to include submicroscopic infections and be implemented in the dry season.

Malaria transmission-blocking activity in experimental infections of Anopheles gambiae from naturally infected Plasmodium falciparum gametocyte carriers.

Experimental infections of anopheline mosquitoes were carried out with Plasmodium falciparum gametocytes from 65 naturally infected patients in Cameroon. A comparison was made between infections with blood containing autologous plasma and blood in which the plasma was replaced with plasma from a donor without previous malaria exposure. A lower infection rate was observed in 50 of 65 autologous plasma samples. Transmission was significantly blocked in 3 infections. This indicates that, in a population living in an area endemic for malaria, blood plasma factor(s) can reduce the transmission capacity of gametocyte carriers to mosquitoes.

Stage-specific effects of host plasma factors on the early sporogony of autologous Plasmodium falciparum isolates within Anopheles gambiae

Quantitatively assessing the impact of naturally occurring transmission‐blocking (TB) immunity on malaria parasite sporogonic development may provide a useful interpretation of the underlying mechanisms. Here, we compare the effects of plasma derived from 23 naturally infected gametocyte carriers (OWN) with plasma from donors without previous malaria exposure (AB) on the early sporogonic development of Plasmodium falciparum in Anopheles gambiae. Reduced parasite development efficiency was associated with mosquitoes taking a blood meal mixed with the gametocyte carriers’ own plasma, whereas replacing autologous plasma with non‐immune resulted in the highest level of parasite survival. Seven days after an infective blood meal, 39.1% of the gametocyte carriers’ plasma tested showed TB activity as only a few macrogametocytes ingested along with immune plasma ended up as ookinetes but subsequent development was blocked in the presence of immune plasma. In other experiments (60.9%), the effective number of parasites declined dramatically from one developmental stage to the next, and resulted in an infection rate that was two‐fold lower in OWN than in AB infection group. These findings are in agreement with those in other reports and go further by quantitatively examining at which transition stages TB immunity exerts its action. The transitions from macrogametocytes to gamete/zygote and from gamete/zygote to ookinete were identified as main targets. However, the net contribution of host plasma factors to these interstage parasite reductions was low (5–20%), suggesting that irrespective of the host plasma factors, mosquito factors might also lower the survival level of parasites during the early sporogonic development.

Transformation of sporozoites of Plasmodium berghei into exoerythrocytic forms in the liver of its mammalian host

SummaryIntrahepatocytic transformation in vivo of the rodent malaria sporozoite of Plasmodium berghei, into the young trophic exoerythrocytic tissue stage was studied by immunofluorescence, light- and electron microscopy. The first 20 h of intracellular life were involved entirely in dedifferentiation with limited proliferation of organelles. From about 20 h onwards nuclear division commenced, rough endoplasmic reticulum became markedly expanded, and mitochondria increased in numbers. However, remains of the sporozoite pellicle (i.e., inner membranes and subpellicular microtubules) persisted for at least 28 h, which correlates with the persisting reaction of young exoerythrocytic forms with antisporozoite antibodies. In general, the basic mechanism of transformation resembles that of the ookinete into oocyst and that of the merozoite into erythrocytic trophozoite.