Effects of the kdr resistance mutation on the susceptibility of wild Anopheles gambiae populations to Plasmodium falciparum: a hindrance for vector control.

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.

Comparative susceptibility to Plasmodium falciparum of the molecular forms M and S of Anopheles gambiae and Anopheles arabiensis.

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.

Dynamics of transmission of Plasmodium falciparum by Anopheles arabiensis and the molecular forms M and S of Anopheles gambiae in Dielmo, Senegal.

BACKGROUND: The adaptation of Anopheles gambiae to humans and its environment involves an ongoing speciation process that can be best demonstrated by the existence of various chromosomal forms adapted to different environments and of two molecular forms known as incipient taxonomic units. METHODS: The aim of this study was to compare the epidemiologic role of Anopheles arabiens is and the molecular forms M and S of Anopheles gambiae in the transmission of Plasmodium in a rural areas of southern Senegal, Dielmo. The sampling of mosquitoes was carried out monthly between July and December 2004, during the rainy season, by human volunteers and pyrethrum spray catches. RESULTS: Anopheles arabiensis, An. gambiae M and S forms coexisted during the rainy season with a predominance of the M form in September and the peak of density being observed in August for the S form. Similar parity rates were observed in An. arabiensis [70.9%] (n = 86), An. gambiae M form [68.7%] (n = 64) and An. gambiae S form [81.1%] (n = 156). The circumsporozoite protein (CSP) rates were 2.82% (n = 177), 3.17% (n = 315) and 3.45% (n = 405), with the mean anthropophilic rates being 71.4% (n = 14), 86.3% (n = 22) and 91.6% (n = 24) respectively for An. arabiensis and An. gambiae M and S forms. No significant difference was observed either in host preference or in Plasmodium falciparum infection rates between sympatric M and S populations. CONCLUSION: No difference was observed either in host preference or in Plasmodium falciparum infection rates between sympatric M and S populations, but they present different dynamics of population. These variations are probably attributable to different breeding conditions.

Age-structured gametocyte allocation links immunity to epidemiology in malaria parasites.

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.

Plasmodium falciparum genotype population dynamics in asymptomatic children from Senegal.

In areas where malaria is endemic, infected individuals generally harbor a mixture of genetically distinct Plasmodium falciparum parasite populations. For the first time, we studied temporal variations of blood parasite densities and circulating genotypes in asymptomatic Senegalese children, at time intervals as short as 4-12 h. Twenty-one Senegalese children, presenting with an asymptomatic P. falciparum infection, were sampled eight times within three days. Parasite density was assessed by thick blood smears, and all infecting genotypes were quantified by the fragment-analysis method. Parasite densities showed dramatic fluctuations up to a 1 to 1,000 ratio, with at least one peak of parasite density. Polyclonal infections were detected in all children, with a multiplicity of infection of 5.2-6.8 genotypes per child. A single sample never reflected the full complexity of the parasite populations hosted by a given individual. Genotypes with different behaviors were detected in all children, some genotypes undergoing major fluctuations, while others were highly stable during the follow-up. A single peripheral blood sampling does not reflect the total parasite load. Repeated sampling is needed to have a more detailed scheme of parasite population dynamics and a better knowledge of the true complexity of an infection.

Identification and typing of Cameroonian isolates of P. malariae using monoclonal antibodies against P. brasilianum.

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 in three areas with perennial or seasonal endemicity and different levels of transmission.

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%).

Immune response of Anopheles gambiae to the early sporogonic stages of the human malaria parasite Plasmodium falciparum.

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.