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International audience; 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.

In the present study, monoclonal antibodies raised against Plasmodium brasilianum were used to demonstrate, for the first time, antigenic diversity in natural populations of Plasmodium malariae isolates and as diagnostic tool to detect low parasitaemia P. malariae infection. Seventeen McAbs reacting by indirect immunoflorescence antibody (IFA) assay with no other Plasmodium species than P. brasilianum, were shown to react with P. malariae and were used for typing 29 P. malariae isolates from hyperendemic areas in Yaounde and in three villages of South Cameroon with parasitaemia ranging from 0.01% to 1.8%. All 29 isolates were distinguished by their ability to react with certain antibodies and considered as representing different isolates of P. malariae. One of these McAbs (No. 14) recognized P. malariae isolates to both in Yaounde and from Mengang but not in Edou or in Nkol Mvae, which may recognize a specific epitope that is less common in strains found in these villages and provide evidence of regional variation within the P. malariae parasites. The McAbs Nos. 16 and 17 were used to determine their usefulness as diagnostic tools for 30 suspected blood samples that were collected from patients with fever and it became clear that they could detect sub-microscopical infections of P. malariae. This study supports the concept of using of P. brasilianum as a substitute for P. malariae during immuno-diagnosis of malaria in endemic areas where PCR assay cannot be used for identification of the P. malariae parasites. In addition our results for the first time provide evidence of considerable antigenic diversity of clinical P. malariae isolates in Cameroon.

Plasmodium falciparum transmission blocking immunity (TBI) was investigated in 3 different endemic areas. Reared Anopheles gambiae s.s. were experimentally infected with the blood of gametocyte carriers, either in the presence of autologous plasma (OWN) or after replacement of the OWN plasma with a nonimmune serum of AB blood group (control). Transmission reduction was defined by a lower level of mosquito infection in the OWN batch compared with the control. After controlling for the effect of gametocytemia, the proportion of "transmission reducers" was lower in the town of Yaounde in Cameroon (UC), (14%, N = 75) than in the two rural areas of South Cameroon (RC) (29%, N = 31) and Sénégal (RS) (44%, N = 32). The contribution of TBI relative to the total inhibition of the parasite development (including human, parasite, and mosquito factors) was higher in RS (49.6%) than in RC (12.6%) and UC (9.5%).

Contains fulltext : 58663.pdf (Publisher’s version ) (Closed access) 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.

Deciphering molecular interactions between the malaria parasite and its mosquito vector is an emerging area of research that will be greatly facilitated by the recent sequencing of the genomes of Anopheles gambiae mosquito and of various Plasmodium species. So far, most such studies have focused on Plasmodium berghei, a parasite species that infects rodents and is more amenable to studies. Here, we analysed the expression pattern of nine An.gambiae genes involved in immune surveillance during development of the human malaria parasite P.falciparum in mosquitoes fed on parasite-containing blood from patients in Cameroon. We found that P.falciparum ingestion triggers a midgut-associated, as well as a systemic, response in the mosquito, with three genes, NOS, defensin and GNBP, being regulated by ingestion of gametocytes, the infectious stage of the parasite. Surprisingly, we found a different pattern of expression of these genes in the An.gambiae–P.berghei model. Therefore, differences in mosquito reaction against various Plasmodium species may exist, which stresses the need to validate the main conclusions suggested by the P.berghei–An.gambiae model in the P.falciparum–An.gambiae system.

Background In the context of generalization of insecticide resistance, the hypothesis that insecticide resistance has a positive impact on the capacity of mosquitoes to transmit malaria constitutes a hindrance for malaria elimination. The aim of this study was to investigated populations of Anopheles coluzzii and Anopheles gambiae S molecular form to assess whether different genotypes at the kdr locus are responsible for different susceptibility to Plasmodium falciparum infection. Methods F3 progeny of An. gambiae s.l. collected in Dielmo were infected by direct membrane feeding with P. falciparum gametocyte-containing blood sampled from volunteer patients. The presence of oocysts was determined by light microscopy after seven days, and the presence of sporozoites by ELISA after 14 days. Mosquito species and molecular forms were identified by PCR. Generalized linear models were performed using the R software to test the effect of explanatory variables including the genotype at the kdr locus on infection rate and density. Results The odds of being infected with oocysts and sporozoites were greater in RS and RR groups than in SS groups (χ2 = 42.8, df = 1, P(>χ2) = 6.1e-11). The density of infection was also dependent on genotype, with RR and RS genotypes showing denser infection than SS genotypes. Pairwise comparisons of oocyst number and absorbance indicated sometime a small betwen species (i.e. between An. gambiae S form, and An. coluzzii), but the effect of genotype was much more important. Conclusion The presence of the resistance allele at the kdr locus increases susceptibility to Plasmodium not only at the oocyst stage but also at the sporozoite stage in non-genetically modified wild mosquitoes. These results have significant implications and should be taken into account in the development of strategies for malaria control.

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.

Background The different taxa belonging to Anopheles gambiae complex display phenotypic differences that may impact their contribution to malaria transmission. More specifically, their susceptibility to infection, resulting from a co-evolution between parasite and vector, might be different. The aim of this study was to compare the susceptibility of M and S molecular forms of Anopheles gambiae and Anopheles arabiensis to infection by Plasmodium falciparum. Methods F3 progenies of Anopheles gambiae s.l. collected in Senegal were infected, using direct membrane feeding, with P. falciparum gametocyte-containing blood sampled on volunteer patients. The presence of oocysts was determined by light microscopy after 7 days, and the presence of sporozoite by ELISA after 14 days. Mosquito species and molecular forms were identified by PCR. Results The oocyst rate was significantly higher in the molecular S form (79.07%) than in the M form (57.81%, Fisher's exact test p < 0.001) and in Anopheles arabiensis (55.38%, Fisher's exact test vs. S group p < 0.001). Mean ± s.e.m. number of oocyst was greater in the An. gambiae S form (1.72 ± 0.26) than in the An. gambiae M form (0.64 ± 0.04, p < 0.0001) and in the An. arabiensis group (0.58 ± 0.04, vs. S group, p < 0.0001). Sporozoite rate was also higher in the molecular form S (83.52%) than in form M (50.98%, Fisher's exact test p < 0.001) and Anopheles arabiensis 50.85%, Fisher's exact test vs. S group p < 0.001). Conclusion Infected in the same experimental conditions, the molecular form S of An. gambiae is more susceptible to infection by P. falciparum than the molecular form M of An. gambiae and An. arabiensis.

A longitudinal study, including entomological, parasitological, immunological and clinical data, was carried out in a rice field and a savanna village in Burkina Faso. In this study, the authors followed the evolution of several parasitological parameters in order to compare the level of immunoprotection in children of these two areas. In particular, the percentages of recently ‘infected’ or ‘recovered’ children were calculated, during the interval separating two consecutive surveys. In both areas, parasite densities quickly increased in children from 0 to 14 years old, immediately after the beginning of the transmission period. In savanna, during the rainy season (May–October), parasite densities increased and the proportion of recently ‘recovered’ children from 0 to 4 years old (becoming parasitologically negative between two consecutive surveys) was very low. On the other hand, parasite densities decreased and the recovery rate was higher in children from 10 to 14 years old before the end of the rainy season, while the transmission was going on. In the rice field area, Plasmodium falciparum densities decreased only at the end of the transmission period (December) and had the same levels as those found in savanna, in spite of a lower inoculation rate. The second peak of transmission seemed neither to increase the proportion of recovered children, nor to boost the immunoprotection of these children.

A survey involving 77 individuals living in two savannah villages near Bobo Dioulasso (Burkina Faso, West Africa), was performed in June 1987 (before), August-September (during) and January 1988 after the seasonal transmission. The clinical longitudinal study during the seasonal period permitted us to define three different groups in terms of both age and occurrence of malaria attack (MA; greater than or equal to 5000 parasites/mm3 of blood and axillary fever greater than or equal to 37.8 degrees C). The presence of circulating stable antigen (S-Ag) and the antibody responses against exoantigens (E-Ag) of Plasmodium falciparum were also evaluated at three observations periods: beginning, during and after the transmission season. The adult group (III) had the highest rates of IgG Ab to E-Ag although, IgM prevalence to E-Ag was maximal in the group II (individuals with no malaria attack and age less than or equal to 15 years old). Group I (persons with less than or equal to 15 years old and who contracted at least one MA) did not have any S-Ag at the first observation period and showed the lowest rate of antibodies to E-Ag. The probability of occurrence of an MA calculated from these parameters at the beginning of the transmission period were correct in 78.9% of the cases in children (Groups I & II) and in 71.8% of adults during the subsequent transmission period. Therefore these values could be used for evaluating the probability of occurrence of a clinical MA during the transmission period in a mesoendemic area. S-Ag and antibodies to E-Ag could participate positively in the mechanisms involved in the development of the immune status.

The plasma levels of tumor necrosis factor were measured during a longitudinal survey of 84 subjects living in an endemic area of malaria. In most cases, the plasma tumor necrosis factor was found at its highest level during the malaria transmission peak and became normal again during the dry season. Children having suffered from malarial attack keep low tumor necrosis factor levels compared to adults and asymptomatic children. These results suggest that tumor necrosis factor could be associated with the development of resistance against malaria.

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.

Background Despite a long history of attempts to model malaria epidemiology, the over-riding conclusion is that a detailed understanding of host-parasite interactions leading to immunity is required. It is still not known what governs the duration of an infection and how within-human parasite dynamics relate to malaria epidemiology. Presentation of the hypothesis Immunity to Plasmodium falciparum develops slowly and requires repeated exposure to the parasite, which thus generates age-structure in the host-parasite interaction. An age-structured degree of immunity would present the parasite with humans of highly variable quality. Evolutionary theory suggests that natural selection will mould adaptive phenotypes that are more precise (less variant) in "high quality" habitats, where lifetime reproductive success is best. Variability in malaria parasite gametocyte density is predicted to be less variable in those age groups who best infect mosquitoes. Thus, the extent to which variation in gametocyte density is a simple parasite phenotype reflecting the complex within-host parasite dynamics is addressed. Testing the hypothesis Gametocyte densities and corresponding infectiousness to mosquitoes from published data sets and studies in both rural and urban Cameroon are analysed. The mean and variation in gametocyte density according to age group are considered and compared with transmission success (proportion of mosquitoes infected). Across a wide range of settings endemic for malaria, the age group that infected most mosquitoes had the least variation in gametocyte density, i.e. there was a significant relationship between the variance rather than the mean gametocyte density and age-specific parasite transmission success. In these settings, the acquisition of immunity over time was evident as a decrease in asexual parasite densities with age. By contrast, in an urban setting, there were no such age-structured relationships either with variation in gametocyte density or asexual parasite density. Implications of the hypothesis Gametocyte production is seemingly predicted by evolutionary theory, insofar as a reproductive phenotype (gametocyte density) is most precisely expressed (i.e. is most invariant) in the most infectious human age group. This human age group would thus be expected to be the habitat most suitable for the parasite. Comprehension of the immuno-epidemiology of malaria, a requisite for any vaccine strategies, remains poor. Immunological characterization of the human population stratified by parasite gametocyte allocation would be a step forward in identifying the salient immunological pathways of what makes a human a good habitat.

SUMMARYThis study investigated the significance of serum complement on transmission-reducing activity (TRA) of field sera from 24 infectedPlasmodium falciparumgametocyte carriers (from Cameroon) against cultured NF54P. falciparum.Laboratory-rearedAnopheles stephensiwere 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 onP. falciparumare consistent with previous studies on animal hosts ofPlasmodium, indicating that earlyP. falciparumsporogonic stages may be insensitive to the antibody-dependent pathways of complement in human serum.

Contains fulltext : 57970.pdf (Publisher’s version ) (Closed access) 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.

International audience; 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.

International audience; 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.

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.

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. (Résumé d'auteur)

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.

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.

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 postinfection. 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.

Neopterin levels in plasma were measured during a longitudinal study of 75 patients living in an area endemic for malaria. The maximum levels were observed in August/September, the period of peak transmission of the disease. During the dry season, levels remain elevated. No relationship was found between the level of neopterin and the individual's immune status against malaria. Tumor necrosis factor (TNF), activated T lymphocytes subpopulations, solable interleukin 2 receptors, and anti-circumsporozoite protein antibodies were not correlated with neopterin levels during the period of peak transmission. In contrast, neopterin levels were correlated with anti- Plasmodium falciparum antibodies detected by immunofluorescence. It was concluded that, in a population of chronic parasite carriers subjected to repeated infestations, neopterin was a good indicator of slowly acquired immunity in stable transmission area, but it poorly reflected acute immune responses.

The malarial infectivity ofan African village population was tested by selecting a demographically representative sample of individuals for study, regardless of parasitemia or gametocytemia. The infectivity of this population people to laboratory-bred mosquitoes was investigated using membrane feeding techniques. Tests on 322 subjects (greater than four years ofage) indicated that approximately 48.4% were capable ofinfecting mosquitoes. There were similar proportions of infectious individuals among gametocyte carriers (52.5%) and nongametocyte carriers (46.6%). All age groups appeared to contribute equally to this infective reservoir. Most of the infections resulted in low oocyst loads (1.8 oocysts) on the midgut of the positive mosquitoes and only a few mosquitoes per batch were infected (1 1.5%). A previous entomologic survey estimated 90 infected bites/person/year and a low parity index in Anopheles gainbiae (< 60%) as well as in An. fiinestus (< do%), the two main malaria vectors in this region. This low panty index could indicate a low life expec- tancy for infected mosquitoes and could therefore explain an inoculation rate lower than expected considering the high degree of infectivity of the human population studied. An estimation of the infectiousness of Plas- modium falciparum gametocyte carriers to mos- quitoes is of both theoretical and practical in- terest for understanding the epidemiology of malaria and its changes after application of con- trol measures. There are many ways to estima?e this infectiousness. A direct approach is to feed batches of mosquitoes on a demographically rep- resentative human population, regardless of par- asitemia or gametocytemia, either by membrane feeding,'.* or by feeding laboratory-bred mos- quitoes directly on the skin of individuals.' An- other approach is to determine the mosquito in- fection pr~bability.~ A third is to measure the age-specific sporozoite rates in anopheline vec- tor~.~ In this report, we have used the first approach, which has only been applied one other time in Africa, 30 years ago.' This allowed us to estimate the proportion of infectious individuals in an African village population near Bobo-Dioulasso in southwestern Burkina Faso.

For a better understanding of the epidemiology of Plasmodium falciparum in an African savanna area, the authors have: (a) defined the real gametocyte reservoir in the native population; (b) followed the fluctuations of gametocytaemia throughout the transmission period; and (c) measured the infectiousness of malarious individuals to mosquitoes. The transversal surveys, in different villages of this endemic area, have shown that gametocyte carrier rates decreased with age and malaria experience; 10.9% of the whole population were potentially infectious to mosquitoes, and of these 73% were children and only 27% were adults. The longitudinal studies have shown that the P. falciparum gametocyte rate depends on the equilibrium between the gametocyte conversion rates and the density of the asexual forms. When there are large numbers of children who become carriers of the sexual stage of the parasite and at the same time a small number who lose their gametocyte infection, the gametocyte rate increases in the population; and vice versa. The circumstances under which gametocytes are produced are not well-known. Two factors seem to be important: the level of the parasite density and immune mechanisms. The infectiousness of malarious individuals was estimated by the 'mosquito infection probability'. The percentage of mosquitoes infected after feeding on gametocyte carriers (which may partly reflect the infectiousness of a human population to mosquitoes) was multiplied by the percentage of gametocyte carriers in the population. This indicated that, in this endemic area, 4% of biting mosquitoes would become infected; but this theoretical mosquito infection probability is over-estimated.(ABSTRACT TRUNCATED AT 250 WORDS)

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