Role of heat-labile serum factor or host complement in the inhibition of Plasmodium falciparum sporogonic stages in Anopheles stephensi by gametocyte carriers' serological factors.

This study investigated the significance of serum complement on transmission-reducing activity (TRA) of field sera from 24 infected Plasmodium falciparum gametocyte carriers (from Cameroon) against cultured NF54 P. falciparum. Laboratory-reared Anopheles stephensi were given infectious blood meals prepared either with sera from naïve Dutch donor (AB type) or pair-matched field serum samples, both with and without active complement. TRA of serum factors and host complement on mosquito infection rate and oocyst intensity were divided into the various components involved in the early stages of sporogony. The majority (>80%) of sera tested showed positive antibody titres to Pfs230, the relevant complement-dependent target of transmission-reducing mechanisms. Regardless of the presence of active complement, bloodmeals with field sera exhibited significantly lower infection rates and oocyst intensity than the control group. Serological reactivity in Capture-ELISA against Pfs230 was significantly correlated with the reduction of parasite infectivity. Contrary to our expectation, the presence of active complement in the mosquito bloodmeal did not increase parasite losses and therefore the magnitude of transmission reduction by individual immune sera. Our findings on P. falciparum are consistent with previous studies on animal hosts of Plasmodium, indicating that early P. falciparum sporogonic stages may be insensitive to the antibody-dependent pathways of complement in human serum.

Aggregation in malaria parasites places limits on mosquito infection rates.

Gametocytes are responsible for the transmission of malaria parasites, Plasmodium spp., from man to mosquito. Although transmission success, as measured by the proportion of mosquitoes infected, generally increases with gametocyte density, the proportion of parasites that are gametocytes is always paradoxically only a few percent of the asexual blood parasites. To address this paradox, we analyse transmission data sets from an urban and an adjacent rural setting in Cameroon to elucidate whether there are discernable lower and upper limits to Plasmodium falciparum gametocyte density that are linked to transmission success. We find that there exists a lower gametocyte density at which mosquito infection rates considerably increase. In addition, we identify upper gametocyte densities at which mosquito infection rates level off. Greatest increases in infection rates occur at low gametocyte densities and coincide with maximum oocyst aggregation within the infected mosquito population. This aggregated oocyst distribution remains despite increases in gametocyte density and ever-decreasing gains in mosquito infection rates. There is increasing suggestion that malaria parasites have evolved sex allocation strategies to ensure transmission in response to a changing, transmission-blocking environment. Here transmission-blocking immunity is proposed not only to ensure low density gametocyte transmission success but also to impose upper limits on transmission success.

Carboxypeptidases B of Anopheles gambiae as targets for a Plasmodium falciparum transmission-blocking vaccine.

Anopheles gambiae is the major African vector of Plasmodium falciparum, the most deadly species of human malaria parasite and the most prevalent in Africa. Several strategies are being developed to limit the global impact of malaria via reducing transmission rates, among which are transmission-blocking vaccines (TBVs), which induce in the vertebrate host the production of antibodies that inhibit parasite development in the mosquito midgut. So far, the most promising components of a TBV are parasite-derived antigens, although targeting critical mosquito components might also successfully block development of the parasite in its vector. We previously identified A. gambiae genes whose expression was modified in P. falciparum-infected mosquitoes, including one midgut carboxypeptidase gene, cpbAg1. Here we show that P. falciparum up-regulates the expression of cpbAg1 and of a second midgut carboxypeptidase gene, cpbAg2, and that this up-regulation correlates with an increased carboxypeptidase B (CPB) activity at a time when parasites establish infection in the mosquito midgut. The addition of antibodies directed against CPBAg1 to a P. falciparum-containing blood meal inhibited CPB activity and blocked parasite development in the mosquito midgut. Furthermore, the development of the rodent parasite Plasmodium berghei was significantly reduced in mosquitoes fed on infected mice that had been immunized with recombinant CPBAg1. Lastly, mosquitoes fed on anti-CPBAg1 antibodies exhibited reduced reproductive capacity, a secondary effect of a CPB-based TBV that could likely contribute to reducing Plasmodium transmission. These results indicate that A. gambiae CPBs could constitute targets for a TBV that is based upon mosquito molecules.

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

Summary 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.

Plasmodium falciparum transmission blocking immunity under conditions of low and high endemicity in Cameroon.

Transmission blocking immunity (TBI) was studied in relation to age, gametocyte density and transmission intensity. subjects with high gametocytaemias were selected in a hypo-endemic urban district and a hyper-endemic rural area in South Cameroon. TBI was determined in blood from gametocyte carriers in a bioassay (Direct Membrane Feeding Assay), with either autologous plasma (OWN) or control serum (AB). Mosquito infection rates (IR) were compared. infection rates correlated positively with gametocyte and oocyst densities. Three TBI indicators were analysed: the proportion of transmission reducers (IRAB > IROWN, P < 0.01), the mean intensity of TBI (IRAB - IROWN), and the contribution of TBI to total inhibition [(IRAB-IROWN)/(100-IROWN)]. we could not discriminate between areas with regard to either the proportion of transmission reducers (urban 15% and rural 29%) or the mean levels of TBI (urban 10% and rural 9%), or contribution of TBI to total inhibition (urban 10% and rural 13%). there was no relationship between TBI indicators and age, but a trend of increasing values was observed with rising gametocytaemia, which was considered as a confusing factor. a multivariable analysis showed that the probability of being a reducer was 4.6 fold higher in the rural area than in the urban district.

Togetherness among Plasmodium falciparum gametocytes: interpretation through simulation and consequences for malaria transmission.

Previous experimental gametocyte infections of Anopheles arabiensis on 3 volunteers naturally infected with Plasmodium falciparum were conducted in Senegal. They showed that gametocyte counts in the mosquitoes are, like macroparasite intakes, heterogeneous (overdispersed). They followed a negative binomial distribution, the overdispersion coefficient seeming constant (k = 3.1). To try to explain this heterogeneity, we used an individual-based model (IBM), simulating the behaviour of gametocytes in the human blood circulation and their ingestion by mosquitoes. The hypothesis was that there exists a clustering of the gametocytes in the capillaries. From a series of simulations, in the case of clustering the following results were obtained: (i) the distribution of the gametocytes ingested by the mosquitoes followed a negative binomial, (ii) the k coefficient significantly increased with the density of circulating gametocytes. To validate this model result, 2 more experiments were conducted in Cameroon. Pooled experiments showed a distinct density dependency of the k-values. The simulation results and the experimental results were thus in agreement and suggested that an aggregation process at the microscopic level might produce the density-dependent overdispersion at the macroscopic level. Simulations also suggested that the clustering of gametocytes might facilitate fertilization of gametes.

Estimation of malaria transmission from humans to mosquitoes in two neighbouring villages in south Cameroon: evaluation and comparison of several indices.

Malaria transmission from humans to mosquitoes was assessed in two neighbouring villages in a rural area near Yaoundé, Cameroon during high and low transmission seasons during 1998-2000, using several indices previously evaluated in different areas endemic for malaria but never directly compared. These indices were estimated from human parasitological data and mosquito infection rates and, for each individual, thick blood films were prepared at the same time as experimental infection of laboratory-bred mosquitoes. Among the 685 volunteers examined, the prevalence of Plasmodium falciparum gametocyte carriers was 16%, and 8% of individuals were able to infect mosquitoes. The percentage of mosquitoes that became infected by feeding on the infectious individuals was 21%. Children aged < 10 years contributed to about 75% of the infectious reservoir, although they constituted only 35% of the total population. Differences were found between the transmission seasons and the villages, and varied according to the index examined. Although there were more infectious individuals in one of the two villages, they were less infectious than those in the other village during the high transmission season. Comparative analysis of the transmission indices suggests the existence of functioning transmission-blocking immunity in one of the villages, which until now has been only hypothetically considered to play a role in malaria transmission in a natural setting. The epidemiological value of all the indices used and their accuracy in estimating the human infectious reservoir and its natural or induced variations are discussed.

[Biology of man-mosquito Plasmodium transmission]. / Biologie de la transmission homme-moustique du Plasmodium.

The transmission of Plasmodium is a complex process that requires the encounter of three organisms: the parasite, the host and the mosquito vector. For a better understanding, the transmission of this parasite can be divided in four cyclical steps: 1) the development in man, including gametocytogenesis, 2) the transfer from man to mosquito, 3) the sporogonic development in the mosquito, 4) the transfer from mosquito to man. The present article proposes a review of these different aspects and focuses especially to recent biological knowledge on Plasmodium falciparum in the gametocytogenesis issues, infectivity of gametocytes, sporogonic development and natural factors limiting this development.

[Plasmodium falciparum: epidemiology and man-mosquito transmission and infection in the vector]. / Plasmodium falciparum: épidémiométrie de la transmission homme-moustique et de l'infection chez le vecteur.

The level of malaria transmission is usually estimated by some entomological parameters (entomological inoculation rate or reproductive rate and its seasonal variations). However, only one aspect of the malaria transmission is explored by this way, i.e. the transmission of Plasmodium from mosquito to man. The transmission from man to mosquito, the development of parasite in the mosquito midgut, and the role of transmission blocking immunity remain poorly documented. Recent studies on vaccination with gamete antigens showed that transmission blocking immunity, and the natural infectiousness of gametocytes after treatment underlined the need for taking into account a new aspect of malaria epidemiology concerning the transmission of Plasmodium from man to mosquito. In this paper, authors propose and discuss different new indicators and methods to improve our knowledge on malaria epidemiology.

Level and dynamics of malaria transmission and morbidity in an equatorial area of South Cameroon.

We conducted parasitological and entomological malaria surveys among the population of Mengang district in southern Cameroon to analyse the relationship between malaria transmission intensity and malaria morbidity. We investigated two adjacent areas which differ 10-fold in transmission intensity [annual entomological inoculation rate (EIR) 17 vs. 170], but have very similar Plasmodium falciparum malariometric profiles with parasite prevalences of 58 vs. 64%, high parasitaemia prevalences (> 1000 parasites/microl) of 15 vs. 16% and the same morbidity of 0.17-0.5 attacks/person/year. Plasmodium malariae prevalence was 14 vs. 16%. One possible explanation is that the similarity of the duration of the short and high transmission seasons in both areas is equally, if not more, significant for parasitological and clinical profiles as the annual EIR. We discuss the relationships between variations in transmission levels, parasitaemia and clinical incidence, and draw parallels to similar situations elsewhere.

Transcripts of the malaria vector Anopheles gambiae that are differentially regulated in the midgut upon exposure to invasive stages of Plasmodium falciparum.

Understanding the interactions between the most deadly malaria parasite, Plasmodium falciparum, and its main vector, Anopheles gambiae, would be of great help in developing new malaria control strategies. The malaria parasite undergoes several developmental transitions in the mosquito midgut and suffers population losses to which mosquito factors presumably contribute. To identify such factors, we analysed An. gambiae midgut transcripts whose expression is regulated upon ingestion of invasive or non-invasive forms of P. falciparum using a differential display approach. Sixteen cDNA were studied in detail; 12 represent novel genes of An. gambiae including a gene encoding profilin. Four transcripts were specifically regulated by P. falciparum gametocytes (invasive forms), whereas the others were regulated by either non-invasive or both non-invasive and invasive forms of the parasite. This differential regulation of some genes may reflect the adaptation of P. falciparum to its natural vector. These genes may be involved in the development of P. falciparum in An. gambiae or in the defence reaction of the mosquito midgut towards the parasite.

Cytoadherence of Plasmodium falciparum-infected erythrocytes in the human placenta.

In Plasmodium falciparum-parasitized pregnant women, erythrocytes infected by mature stages of the parasite sequester into placental intervillous spaces. The presence of parasites in the placenta causes maternal anaemia and low birth weight of the infant. In-vitro studies suggest placental sequestration may involve the cytoadherence of infected erythrocytes to chondroitin sulphate A (CSA) and/or intercellular adhesion molecule 1 (ICAM-1) expressed by human placental syncytiotrophoblast. We identified P. falciparum receptors expressed on the surface of human syncytiotrophoblast using immunofluorescence of placental biopsies from Cameroon, a malaria-endemic area. In all placentas, a strongly positive staining was observed on the syncytiotrophoblast for CSA, but not for ICAM-1, vascular endothelium cell adhesion molecule-1, E-selectin, nor CD36. The cytoadherence ability of parasites from pregnant women and nonpregnant subjects was assessed on in-vitro cultured syncytiotrophoblast. Parasites from pregnant women bound to the trophoblast via CSA but not ICAM-1. Parasites from nonpregnant hosts either did not bind to the trophoblast culture or bound using ICAM-1. Our data support the idea that placental sequestration may result from cytoadherence to placental trophoblast and that pregnant women are parasitized by parasites that differ from parasites derived from nonpregnant host by their cytoadherence ability.

Comparison of artificial membrane feeding with direct skin feeding to estimate infectiousness of Plasmodium falciparum gametocyte carriers to mosquitoes.

Human infectiousness to mosquitoes can be estimated by 2 tests: direct feeding on the skin and membrane feeding on venous blood. To validate the membrane feeding assay, the infectiousness of Plasmodium falciparum gametocyte carriers to Anopheles gambiae was estimated by these 2 methods in the same individuals in a rural area of Cameroon. Results from 37 experiments showed that direct feeding gave significantly higher infection rates than membrane feeding. We observed an average of 19.4% infected mosquitoes by direct feeding compared with 12.1% by membrane feeding, and a mean oocyst load of 5.63 by direct feeding compared with 2.65 by membrane feeding. However, there was a very good concordance between the 2 tests: 84.3% with the Kappa test on percentages of infected mosquitoes and 98.7% with the interclass correlation coefficient on oocyst loads. In addition, we found a good linear correlation between the 2 methods.

Development of antibodies against chondroitin sulfate A-adherent Plasmodium falciparum in pregnant women.

In areas where Plasmodium falciparum is endemic, pregnant women are at increased risk for malaria, and this risk is greatest during the first pregnancy. The placenta sequesters parasites that are able to cytoadhere to chondroitin sulfate A (CSA), a molecule expressed by the placental syncytiotrophoblast, while parasites from a nonpregnant host do not bind to CSA. Cytoadherence is mediated by the expression of variants of the P. falciparum-erythrocyte membrane protein 1 family. Each member of this molecule family induces antibodies that specifically agglutinate infected erythrocytes and inhibit their cytoadherence ability. We investigated whether the higher susceptibility of primigravidae was related to the lack of immune response towards CSA-binding parasites. In a cross-sectional study, primigravidae delivering with a noninfected placenta were less likely to have antibodies agglutinating CSA-binding parasites than multigravidae (P < 0.01). In contrast, parasites from nonpregnant hosts were as likely to be recognized by the sera from women of various parities. In a longitudinal study, at 6 months of pregnancy, antibodies against CSA-binding parasites were present in 31.8% of primigravidae and in 76.9% of secundigravidae (P = 0.02). The antibodies against CSA-binding parasites inhibited the cytoadherence of a CSA-adherent parasite strain to the human placental trophoblast. Our data support the idea that the higher susceptibility of primiparae is related to a lack of a specific immune response to placental parasites.

Longitudinal study of Plasmodium falciparum infection and immune responses in infants with or without the sickle cell trait.

BACKGROUND: Individuals may be homozygous (SS) or heterozygous (AS) sickle cell gene carriers or have normal adult haemoglobin (AA). Haemoglobin S could have a protective role against malaria but evidence is sparse and the operating mechanisms are poorly known. METHODS: We followed two cohorts of children. The first was enrolled at birth (156 newborn babies) and the second at 24-36 months old (84 children). Both cohorts were followed for 30 months; monthly for parasitological data and half yearly for immunological data. RESULTS: In the first cohort, 22%, and in the second 13% of children were AS. Whatever their age parasite prevalence rates were similar in AA and AS individuals. Mean parasite densities increased less rapidly with age in AS than in AA children, and were significantly lower in AS than in AA children >48 months old. The AA children tended to be more often admitted to hospital than AS children (22% versus 11%, NS). Both anti-Plasmodium falciparum and anti-Pfl55/RESA antibody rates increased more rapidly in AA than in AS children. Conversely, the prevalence rate of cellular responders to the Pfl55/RESA antigen was similar in AA and AS children during the first 2 years of life, then it was higher in AS than in AA children. CONCLUSIONS: Sickle cell trait related antimalarial protection varies with age. The role of the modifications of the specific immune response to P. falciparum in explaining the protection of AS children against malaria is discussed.

The use of anti-Pfs 25 monoclonal antibody for early determination of Plasmodium falciparum oocyst infections in Anopheles gambiae: comparison with the current technique of direct microscopic diagnosis.

Experimental infections of laboratory-reared anopheline mosquitoes were carried out with 57 Plasmodium falciparum gametocyte carriers from Cameroon. Prevalence of infected mosquitoes and oocyst intensity were determined by two independent methods. Young P. falciparum oocysts were detected on day 2 after feeding using an immunofluorescent assay, and the results were compared with direct microscopic examination of midgut oocysts on day 7 postinfection. The immunofluorescent assay was based on a FITC-labeled anti-25-kDa monoclonal antibody, while the direct microscopy was performed on midguts stained with 2% mercurochrome. Young oocysts were easily detected by their typical and bright green-fluorescing Pfs25 positive coat and their characteristic pattern of pigment granules under transmitted white light examination. The agreement between the results of the two methods was assessed using the Kappa coefficient on prevalences of positive infections and the interclass correlation coefficient on arithmetic mean oocyst load per infected midgut. The results indicated a low agreement between the two methods for the comparison of prevalences of infected mosquitoes. However, this agreement was near perfect for the comparison of mean oocyst intensities. Prevalences of positive infections and the overall number of parasites per positive gut were significantly correlated for both methods. Thus, the immunofluorescent test could be an appropriate tool for early determination of malaria infection in mosquitoes, particularly under laboratory conditions. The possible applications of this immuno-fluorescent technique are discussed.

Plasmodium falciparum: membrane feeding assays and competition ELISAs for the measurement of transmission reduction in sera from Cameroon.

The effect of natural malaria transmission-blocking factors in the blood of Plasmodium falciparum gametocyte carriers was assessed in two types of functional bioassays. In the direct membrane feeding assay (DMFA), a comparison is made between the infectivity of gametocytes from a naturally infected gametocyte carrier in the presence of autologous plasma and the infectivity in the presence of replacement plasma from nonimmune donors. In the standard membrane feeder assay (SMFA), cultured NF54 gametocytes are used to measure the capacity of endemic sera to block transmission. In the DMFA, 18 out of 48 sera (37.5%) from Cameroonian gametocyte carriers reduced transmission significantly, while in the SMFA 22 out of 48 sera (45.8%) produced transmission reduction. There was a positive correlation between both assays (r + 0.41, P < 0.05). Antibodies against epitopes of transmission-blocking target antigens Pfs48/45 and Pfs230 were measured in competition ELISAs and compared with the results of DMFA and SMFA. Serological reactivity in competition ELISAs against three epitopes of Pfs48/45 was significantly higher in the group of transmission-reducing sera in both the DMFA and the SMFA, especially for epitope III. No significant difference was found for Pfs230 antibodies (epitope I). Sensitivity of the serological assays was approximately 60%, with a specificity of around 70%. Serological tests cannot replace the functional bioassay in field situations as yet, but can contribute in the selection of sera for SMFA evaluation.

[Malaria transmission in an area of future vaccination in equatorial forest of south Cameroon]. / Etude de la transmission du paludisme dans une future zone d'essai vaccinal en forêt équatoriale du sud Cameroun.

In order to describe malaria transmission in a future antigamete vaccine trial area, a longitudinal entomological study was conducted, together with parasitological and immunological surveys, from June 1997 to May 1998 in two nearby villages in a tropical rain forest area 100 km east of Yaoundé. Koundou is located along the main road in an open and degraded environment combining cultivated lands and forests; Ebolakounou is located 5 km from the road in forest surroundings. Indoor mosquito night catches no human volunteers were performed twice a month, in ten houses. We determined the entomological infection rate as 176 infected bites per human per year in Koundou (47.7% for An. moucheti, 47.3% for An. gambiae and 5% for An. funestus) and only 17.7 infected bites/human/year in Ebolakounou, with An. gambiae only. Transmission appears to be ten times higher in the village situated in a degraded environment than in the village situated in the rainforest.

Plasmodium falciparum-isolates from Cameroonian pregnant women do not rosette.

The placenta of pregnant women is frequently parasitized by erythrocytes infected by mature stages of Plasmodium falciparum (IE), a phenomenon associated with low birth weight of the offspring. The cytoadherence phenotype of the parasites from pregnant women suggests that placental sequestration may result from cytoadherence to the syncytiotrophoblast. However, as anatomopathological studies report that cytoadherence in the placenta is a rare event, we investigated whether placental parasites may sequester by forming rosettes with uninfected erythrocytes, another possible sequestration mechanism. Parasites from placental blood as well as parasites from the peripheral blood of pregnant and non pregnant subjects were assessed for their ability to rosette. In non pregnant subjects, the rosetting capacity of parasites was as reported in literature while, except in one case, parasites from pregnant women did not rosette. We conclude that the lack of rosetting is a new feature of IEs from pregnant women and that rosetting cannot be involved in the placental sequestration of IEs.

[Levels of malaria transmission: methods and parameters]. / L'évaluation des niveaux de transmission palustre: méthodologies et paramètres.

Evaluation of malaria transmission levels is necessary to compare ecologically diverse areas and to assess the effectiveness of efforts to control the disease. The purpose of this report is to describe useful techniques for descriptive epidemiology and potentially pertinent indicators regarding the three links in the epidemiological chain: transmission from mosquito to man, transmission from man to mosquito, and sporogonic cycle. Standards for evaluation of transmission from mosquito to man are now well established. Techniques and resulting data, mostly entomological, have been validated in numerous multicenter and multidiscipline studies before and after implementation of control measurements. Evaluation of transmission from man to mosquito has not yet been extensively studied. Gametocyte index does not appear to be a good indicator of infectivity in mosquitoes. Two other parameters that have been proposed in the literature are rate of human infectivity to mosquitoes and probability that a bloodmeal will be infectious. However these evaluation techniques have been neither subjected to comparative study nor validated in epidemiological surveys. The third factor for evaluation of malaria transmission levels involves sexual development of the gametocytes (sporogonic cycle) in the vector. Two indicators that might be useful in this regard are quantification of early-stage parasites in the stomach of the mosquito and study of blood factors in subjects in whom inhibition transmission has been documented. Since these methods have been used only sporadically, further study will be needed to validate this approach to evaluate transmission level.