Do the venous blood samples replicate malaria parasite densities found in capillary blood? A field study performed in naturally-infected asymptomatic children in Cameroon.

BACKGROUND: The measure of new drug- or vaccine-based approaches for malaria control is based on direct membrane feeding assays (DMFAs) where gametocyte-infected blood samples are offered to mosquitoes through an artificial feeder system. Gametocyte donors are identified by the microscopic detection and quantification of malaria blood stages on blood films prepared using either capillary or venous blood. However, parasites are known to sequester in the microvasculature and this phenomenon may alter accurate detection of parasites in blood films. The blood source may then impact the success of mosquito feeding experiments and investigations are needed for the implementation of DMFAs under natural conditions. METHODS: Thick blood smears were prepared from blood obtained from asymptomatic children attending primary schools in the vicinity of Mfou (Cameroon) over four transmission seasons. Parasite densities were determined microscopically from capillary and venous blood for 137 naturally-infected gametocyte carriers. The effect of the blood source on gametocyte and asexual stage densities was then assessed by fitting cumulative link mixed models (CLMM). DMFAs were performed to compare the infectiousness of gametocytes from the different blood sources to mosquitoes. RESULTS: Prevalence of Plasmodium falciparum asexual stages among asymptomatic children aged from 4 to 15 years was 51.8% (2116/4087). The overall prevalence of P. falciparum gametocyte carriage was 8.9% and varied from one school to another. No difference in the density of gametocyte and asexual stages was found between capillary and venous blood. Attempts to perform DMFAs with capillary blood failed. CONCLUSIONS: Plasmodium falciparum malaria parasite densities do not differ between capillary and venous blood in asymptomatic subjects for both gametocyte and trophozoite stages. This finding suggests that the blood source should not interfere with transmission efficiency in DMFAs.

Prevalence of Plasmodium falciparum parasites resistant to sulfadoxine/pyrimethamine in pregnant women in Yaoundé, Cameroon: emergence of highly resistant pfdhfr/pfdhps alleles.

OBJECTIVES: To determine, 6 years after the adoption of intermittent preventive treatment of pregnant women with sulfadoxine/pyrimethamine (IPTp-SP) in Cameroon, (i) the polymorphism and prevalence of Plasmodium falciparum dihydrofolate reductase (pfdhfr) and dihydropteroate synthase (pfdhps) gene mutations associated with sulfadoxine/pyrimethamine resistance and (ii) the consequences of sulfadoxine/pyrimethamine use in the selection of pfdhfr/pfdhps alleles. METHODS: pfdhfr and pfdhps genes from P. falciparum isolates collected in Yaoundé (Cameroon) from pregnant women with symptomatic malaria before taking IPTp-SP [SP- group (control) (n = 51)] or afterwards [SP+ group (n = 49)] were sequenced. RESULTS: The pfdhfr N51I, C59R, S108N triple mutant had a prevalence close to 100% (96/100) and no mutations at codons 50 and 164 were detected in either of the groups. The most frequent pfdhps mutation was A437G with a prevalence of 76.5% (39/51) in the SP- group, which was significantly higher in pregnant women who took sulfadoxine/pyrimethamine [95.9% (47/49)] (P = 0.012). Our study confirmed the presence of the pfdhps K540E mutation in Cameroon, but it remained rare. The prevalence of pfdhps A581G and A613S mutations had increased [5.9% (3/51) and 11.8% (6/51) in the control group, respectively] since the last studies in 2005. Surprisingly, the new pfdhps I431V mutation was detected, at a prevalence of 9.8% (5/51), and was found to be associated with other pfdhfr/pfdhps alleles to form an octuple N51I, C59R, S108N/I431V, S436A, A437G, A581G, A613S mutant. CONCLUSIONS: Significant changes were found in pfdhps polymorphism. In particular, we observed several parasites carrying eight mutations in pfdhfr/pfdhps genes, which are very susceptible to having a high level of resistance to sulfadoxine/pyrimethamine.

Midgut microbiota of the malaria mosquito vector Anopheles gambiae and interactions with Plasmodium falciparum infection.

The susceptibility of Anopheles mosquitoes to Plasmodium infections relies on complex interactions between the insect vector and the malaria parasite. A number of studies have shown that the mosquito innate immune responses play an important role in controlling the malaria infection and that the strength of parasite clearance is under genetic control, but little is known about the influence of environmental factors on the transmission success. We present here evidence that the composition of the vector gut microbiota is one of the major components that determine the outcome of mosquito infections. A. gambiae mosquitoes collected in natural breeding sites from Cameroon were experimentally challenged with a wild P. falciparum isolate, and their gut bacterial content was submitted for pyrosequencing analysis. The meta-taxogenomic approach revealed a broader richness of the midgut bacterial flora than previously described. Unexpectedly, the majority of bacterial species were found in only a small proportion of mosquitoes, and only 20 genera were shared by 80% of individuals. We show that observed differences in gut bacterial flora of adult mosquitoes is a result of breeding in distinct sites, suggesting that the native aquatic source where larvae were grown determines the composition of the midgut microbiota. Importantly, the abundance of Enterobacteriaceae in the mosquito midgut correlates significantly with the Plasmodium infection status. This striking relationship highlights the role of natural gut environment in parasite transmission. Deciphering microbe-pathogen interactions offers new perspectives to control disease transmission.

The composition and abundance of bacterial communities residing in the gut of Glossina palpalis palpalis captured in two sites of southern Cameroon.

BACKGROUND: A number of reports have demonstrated the role of insect bacterial flora on their host's physiology and metabolism. The tsetse host and vector of trypanosomes responsible for human sleeping sickness (human African trypanosomiasis, HAT) and nagana in animals (African animal trypanosomiasis, AAT) carry bacteria that influence its diet and immune processes. However, the mechanisms involved in these processes remain poorly documented. This underscores the need for increased research into the bacterial flora composition and structure of tsetse flies. The aim of this study was to identify the diversity and relative abundance of bacterial genera in Glossina palpalis palpalis flies collected in two trypanosomiasis foci in Cameroon. METHODS: Samples of G. p. palpalis which were either negative or naturally trypanosome-positive were collected in two foci located in southern Cameroon (Campo and Bipindi). Using the V3V4 and V4 variable regions of the small subunit of the 16S ribosomal RNA gene, we analyzed the respective bacteriome of the flies' midguts. RESULTS: We identified ten bacterial genera. In addition, we observed that the relative abundance of the obligate endosymbiont Wigglesworthia was highly prominent (around 99%), regardless of the analyzed region. The remaining genera represented approximately 1% of the bacterial flora, and were composed of Salmonella, Spiroplasma, Sphingomonas, Methylobacterium, Acidibacter, Tsukamurella, Serratia, Kluyvera and an unidentified bacterium. The genus Sodalis was present but with a very low abundance. Globally, no statistically significant difference was found between the bacterial compositions of flies from the two foci, and between positive and trypanosome-negative flies. However, Salmonella and Serratia were only described in trypanosome-negative flies, suggesting a potential role for these two bacteria in fly refractoriness to trypanosome infection. In addition, our study showed the V4 region of the small subunit of the 16S ribosomal RNA gene was more efficient than the V3V4 region at describing the totality of the bacterial diversity. CONCLUSIONS: A very large diversity of bacteria was identified with the discovering of species reported to secrete anti-parasitic compounds or to modulate vector competence in other insects. For future studies, the analyses should be enlarged with larger sampling including foci from several countries.

Correction: Application of a qPCR Assay in the Investigation of Susceptibility to Malaria Infection of the M and S Molecular Forms of An. gambiae s.s. in Cameroon.

[This corrects the article DOI: 10.1371/journal.pone.0054820.].

An epidemiologically successful Escherichia coli sequence type modulates Plasmodium falciparum infection in the mosquito midgut.

Malaria transmission relies on the successful development of Plasmodium parasites in the Anopheles mosquito vector. Within the mosquito midgut, malaria parasites encounter a resident bacterial flora and parasite-bacteria interactions modulate Plasmodium development. The mechanisms by which the bacteria interact with malaria parasites are still unknown. The intestinal microbiota could regulate immune signaling pathways or produce bacterial compounds that block Plasmodium development. In this study, we characterized Escherichia coli strains previously isolated from the Anopheles mosquito midgut and investigated the putative role of two E. coli clones, 444ST95 and 351ST73, on parasite development. Sporogonic development was significantly impacted by exposure to clone 444ST95 whereas prevalence and intensity of infection were not different in mosquitoes challenged with 351ST73 as compared to control mosquitoes. This result indicates midgut bacteria exhibit intra-specific variation in their ability to inhibit Plasmodium development. Expression patterns of immune genes differed between mosquitoes challenged with 444ST95 and 351ST73 and examination of the luminal midgut surface by transmission electron microscopy revealed distinct effects of bacterial exposure on midgut epithelial cells. The 444ST95 clone strongly affected mosquito survival and parasite development and this could be associated to the Hemolysin F or other toxins released by the bacteria. Further studies will be needed to decipher the virulence factors and to determine their contribution to the observed phenotype of the 444ST95E. coli strain that belongs to the epidemiological ST95 clonal group responsible for extra intestinal infections in human and other animals.

Impact of exposure to mosquito transmission-blocking antibodies on Plasmodium falciparum population genetic structure.

Progress in malaria control has led to a significant reduction of the malaria burden. Interventions that interrupt transmission are now needed to achieve the elimination goal. Transmission-blocking vaccines (TBV) that aim to prevent mosquito infections represent promising tools and several vaccine candidates targeting different stages of the parasite's lifecycle are currently under development. A mosquito-midgut antigen, the anopheline alanyl aminopeptidase (AnAPN1) is one of the lead TBV candidates; antibodies against AnAPN1 prevent ookinete invasion. In this study, we explored the transmission dynamics of Plasmodium falciparum in mosquitoes fed with anti-AnAPN1 monoclonal antibodies (mAbs) vs. untreated controls, and investigated whether the parasite genetic content affects or is affected by antibody treatment. Exposure to anti-AnAPN1 mAbs was efficient at blocking parasite transmission and the effect was dose-dependent. Genetic analysis revealed a significant sib-mating within P. falciparum infra-populations infecting one host, as measured by the strong correlation between Wright's FIS and multiplicity of infection. Treatments also resulted in significant decrease in FIS as a by-product of drop in infra-population genetic diversity and concomitant increase of apparent panmictic genotyping proportions. Genetic differentiation analyses indicated that mosquitoes fed on a same donor randomly sampled blood-circulating gametocytes. We did not detect trace of selection, as the genetic differentiation between different donors did not decrease with increasing mAb concentration and was not significant between treatments for each gametocyte donor. Thus, there is apparently no specific genotype associated with the loss of diversity under mAb treatment. Finally, the anti-AnAPN1 mAbs were effective at reducing mosquito infection and a vaccine aiming at eliciting anti-AnAPN1 mAbs has a strong potential to decrease the burden of malaria in transmission-blocking interventions without any apparent selective pressure on the parasite population.

Plasmodium falciparum mating patterns and mosquito infectivity of natural isolates of gametocytes.

Plasmodium falciparum infections in malaria endemic areas often harbor multiple clones of parasites. However, the transmission success of the different genotypes within the mosquito vector has remained elusive so far. The genetic diversity of malaria parasites was measured by using microsatellite markers in gametocyte isolates from 125 asymptomatic carriers. For a subset of 49 carriers, the dynamics of co-infecting genotypes was followed until their development within salivary glands. Also, individual oocysts from midguts infected with blood from 9 donors were genotyped to assess mating patterns. Multiplicity of infection (MOI) was high both in gametocyte isolates and sporozoite populations, reaching up to 10 genotypes. Gametocyte isolates with multiple genotypes gave rise to lower infection prevalence and intensity. Fluctuations of genotype number occurred during the development within the mosquito and sub-patent genotypes, not detected in gametocyte isolates, were identified in the vector salivary glands. The inbreeding coefficient Fis was positively correlated to the oocyst loads, suggesting that P. falciparum parasites use different reproductive strategies according to the genotypes present in the gametocyte isolate. The number of parasite clones within an infection affects the transmission success and the mosquito has an important role in maintaining P. falciparum genetic diversity. Our results emphasize the crucial importance of discriminating between the different genotypes within an infection when studying the A. gambiae natural resistance to P. falciparum, and the need to monitor parasite diversity in areas where malaria control interventions are implemented.

Dynamics of Bacterial Community Composition in the Malaria Mosquito's Epithelia.

The Anopheles midgut hosts diverse bacterial communities and represents a complex ecosystem. Several evidences indicate that mosquito midgut microbiota interferes with malaria parasite transmission. However, the bacterial composition of salivary glands and ovaries, two other biologically important tissues, has not been described so far. In this study, we investigated the dynamics of the bacterial communities in the mosquito tissues from emerging mosquitoes until 8 days after a blood meal containing Plasmodium falciparum gametocytes and described the temporal colonization of the mosquito epithelia. Bacterial communities were identified in the midgut, ovaries, and salivary glands of individual mosquitoes using pyrosequencing of the 16S rRNA gene. We found that the mosquito epithelia share a core microbiota, but some bacteria taxa were more associated with one or another tissue at a particular time point. The bacterial composition in the tissues of emerging mosquitoes varied according to the breeding site, indicating that some bacteria are acquired from the environment. Our results revealed temporal variations in the bacterial community structure, possibly as a result of the mosquito physiological changes. The abundance of Serratia significantly correlated with P. falciparum infection both in the midgut and salivary glands of malaria challenged mosquitoes, which suggests that interactions occur between microbes and parasites. These bacteria may represent promising targets for vector control strategies. Overall, this study points out the importance of characterizing bacterial communities in malaria mosquito vectors.

Composition of Anopheles coluzzii and Anopheles gambiae microbiota from larval to adult stages.

During their immature life stages, malaria mosquitoes are exposed to a wide array of microbes and contaminants from the aquatic habitats. Although prior studies have suggested that environmental exposure shapes the microbial community structure in the adult mosquito, most reports have focused on laboratory-based experiments and on a single mosquito epithelium, the gut. In this study, we investigated the influence of the breeding site on the development of the Anopheles coluzzii and Anopheles gambiae microbiota in natural conditions. We characterized bacterial communities from aquatic habitats, at surface microlayer and subsurface water levels, to freshly emerge adult mosquitoes using multiplexed 16S rRNA gene pyrosequencing and we separately analyzed the microbiota associated with the different epithelia of adult individual, midguts, ovaries and salivary glands. We found that the distribution of bacterial communities in the aquatic habitats differed according to the depth of water collections. Inter-individual variation of bacterial composition was large in larvae guts but adult mosquitoes from a same breeding site shared quite similar microbiota. Although some differences in bacterial abundances were highlighted between the different epithelia of freshly emerged An. coluzzii and An. gambiae, an intriguing feature from our study is the particular similarity of the overall bacterial communities. Our results call for further investigations on the bacterial population dynamics in the different tissues to determine the distinctive characteristics of each microbiota during the mosquito lifespan and to identify specific interactions between certain key phyla or species and the insect life history traits.

Anopheles gambiae salivary protein expression modulated by wild Plasmodium falciparum infection: highlighting of new antigenic peptides as candidates of An. gambiae bites.

BACKGROUND: Malaria is the major parasitic disease worldwide caused by Plasmodium infection. The objective of integrated malaria control programs is to decrease malaria transmission, which needs specific tools to be accurately assessed. In areas where the transmission is low or has been substantially reduced, new complementary tools have to be developed to improve surveillance. A recent approach, based on the human antibody response to Anopheles salivary proteins, has been shown to be efficient in evaluating human exposure to Anopheles bites. The aim of the present study was to identify new An. gambiae salivary proteins as potential candidate biomarkers of human exposure to P. falciparum-infective bites. METHODS: Experimental infections of An. gambiae by wild P. falciparum were carried out in semi-field conditions. Then a proteomic approach, combining 2D-DIGE and mass spectrometry, was used to identify the overexpressed salivary proteins in infected salivary glands compared to uninfected An. gambiae controls. Subsequently, a peptide design of each potential candidate was performed in silico and their antigenicity was tested by an epitope-mapping technique using blood from individuals exposed to Anopheles bites. RESULTS: Five salivary proteins (gSG6, gSG1b, TRIO, SG5 and long form D7) were overexpressed in the infected salivary glands. Eighteen peptides were designed from these proteins and were found antigenic in children exposed to the Anopheles bites. Moreover, the results showed that the presence of wild P. falciparum in salivary glands modulates the expression of several salivary proteins and also appeared to induce post-translational modifications. CONCLUSIONS: This study is, to our knowledge, the first that compares the sialome of An. gambiae both infected and not infected by wild P. falciparum, making it possible to mimic the natural conditions of infection. This is a first step toward a better understanding of the close interactions between the parasite and the salivary gland of mosquitoes. In addition, these results open the way to define biomarkers of infective bites of Anopheles, which could, in the future, improve the estimation of malaria transmission and the evaluation of malaria vector control tools.

Application of a qPCR assay in the investigation of susceptibility to malaria infection of the M and S molecular forms of An. gambiae s.s. in Cameroon.

Plasmodium falciparum is the causative agent of malaria, a disease that kills almost one million persons each year, mainly in sub-Saharan Africa. P. falciparum is transmitted to the human host by the bite of an Anopheles female mosquito, and Anopheles gambiae sensus stricto is the most tremendous malaria vector in Africa, widespread throughout the afro-tropical belt. An. gambiae s.s. is subdivided into two distinct molecular forms, namely M and S forms. The two molecular forms are morphologically identical but they are distinct genetically, and differ by their distribution and their ecological preferences. The epidemiological importance of the two molecular forms in malaria transmission has been poorly investigated so far and gave distinct results in different areas. We have developed a real-time quantitative PCR (qPCR) assay, and used it to detect P. falciparum at the oocyst stage in wild An. gambiae s.s. mosquitoes experimentally infected with natural isolates of parasites. Mosquitoes were collected at immature stages in sympatric and allopatric breeding sites and further infected at the adult stage. We next measured the infection prevalence and intensity in female mosquitoes using the qPCR assay and correlated the infection success with the mosquito molecular forms. Our results revealed different prevalence of infection between the M and S molecular forms of An. gambiae s.s. in Cameroon, for both sympatric and allopatric populations of mosquitoes. However, no difference in the infection intensity was observed. Thus, the distribution of the molecular forms of An. gambiae s.s. may impact on the malaria epidemiology, and it will be important to monitor the efficiency of malaria control interventions on the two M and S forms.

Modulation of malaria infection in Anopheles gambiae mosquitoes exposed to natural midgut bacteria.

The development of Plasmodium falciparum within the Anopheles gambiae mosquito relies on complex vector-parasite interactions, however the resident midgut microbiota also plays an important role in mediating parasite infection. In natural conditions, the mosquito microbial flora is diverse, composed of commensal and symbiotic bacteria. We report here the isolation of culturable midgut bacteria from mosquitoes collected in the field in Cameroon and their identification based on the 16S rRNA gene sequencing. We next measured the effect of selected natural bacterial isolates on Plasmodium falciparum infection prevalence and intensity over multiple infectious feedings and found that the bacteria significantly reduced the prevalence and intensity of infection. These results contrast with our previous study where the abundance of Enterobacteriaceae positively correlated with P. falciparum infection (Boissière et al. 2012). The oral infection of bacteria probably led to the disruption of the gut homeostasis and activated immune responses, and this pinpoints the importance of studying microbe-parasite interactions in natural conditions. Our results indicate that the effect of bacterial exposure on P. falciparum infection varies with factors from the parasite and the human host and calls for deeper dissection of these parameters for accurate interpretation of bacterial exposure results in laboratory settings.