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Agroecosystems have been associated with risk of malaria. The aim of this study was to determine the relationship between three agroecosystems: (i) rubber plantation (RP); (ii) oil palm plantation (OPP); (iii) no cash crop plantation (NCCP) and the prevalence of Plasmodium falciparum infection among children living in the Aboisso region. In the three villages within (Ehania-V5) or close (N’zikro) or far from (Ayebo) to each agroecosystem (RP, OPP, and NCCP), two cross-sectional parasitological surveys were carried out during the dry and the peak of the long wet seasons. A total of 586 children aged 1–14 years were recruited in the three villages to determine the prevalence of malaria using conventional microscopy. Plasmodium falciparum was the dominant species with an overall infection prevalence of 40.8%. There was a significant difference in prevalence between agroecosystems, during both the dry (p = 0.002) and wet seasons (p

Background In malaria-endemic areas, human populations are frequently exposed to immunomodulatory salivary components injected during mosquito blood feeding. The consequences on pathogen-specific immune responses are not well known. This study evaluated and compared the humoral responses specific to merozoite stage vaccine candidates of Plasmodium falciparum, in children differentially exposed to Anopheles bites in a natural setting. Methods The cross-sectional study was carried out in Bouaké (Côte d’Ivoire) where entomological data and blood samples from children (0–14 years) were collected in two sites with similar malaria prevalence. Antibody (IgG, IgG1, IgG3) responses to PfAMA1 and PfMSP1 were evaluated by ELISA. Univariate and multivariate analysis were performed to assess the relationship between the immune responses to P. falciparum antigens and exposure to Anopheles bites in the total cohort and in each site, separately. The individual level of exposure to Anopheles bites was evaluated by quantifying specific IgG response to the Anopheles gSG6-P1 salivary peptide, which represents a proxy of Anopheles exposure. Results The anti-Plasmodium humoral responses were different according to the level of exposure of children, with those highly exposed to Anopheles presenting significantly lower antibody responses to PfMSP1 in total population (IgG and IgG3) and in Petessou village (IgG, IgG1, IgG3). No significant difference was seen for PfAMA1 antigen between children differently exposed to Anopheles. In Dar-es-Salam, a neighbourhood where a high Culex density was reported, children presented very low antibody levels specific to both antigens, and no difference according to the exposure to Anopheles bites was found. Conclusion These findings may suggest that immunomodulatory components of Anopheles saliva, in addition to other factors, may participate to the modulation of the humoral response specific to Plasmodium merozoite stage antigens. This epidemiological observation may form a starting point for additional work to decipher the role of mosquito saliva on the modulation of the anti-Plasmodium acquired immunity and clinical protection in combining both field and ex vivo immunological studies.

Background Culex mosquitoes are vectors for a variety of pathogens of public health concern. New indicators of exposure to Culex bites are needed to evaluate the risk of transmission of associated pathogens and to assess the efficacy of vector control strategies. An alternative to entomological indices is the serological measure of antibodies specific to mosquito salivary antigens. This study investigated whether the human IgG response to both the salivary gland extract and the 30 kDa salivary protein of Culex quinquefasciatus may represent a proxy of human exposure to Culex bites. Methodology/Principal findings A multidisciplinary survey was conducted with children aged 1 to 14 years living in neighborhoods with varying exposure to Culex quinquefasciatus in the city of Bouaké, Côte d’Ivoire. Children living in sites with high exposure to Cx quinquefasciatus had a significantly higher IgG response to both salivary antigens compared with children living in the control site where only very few Culex were recorded. Moreover, children from any Culex-high exposed sites had significantly higher IgG responses only to the salivary gland extract compared with children from the control village, whereas no difference was noted in the anti-30 kDa IgG response. No significant differences were noted in the specific IgG responses between age and gender. Sites and the use of a bed net were associated with the level of IgG response to the salivary gland extract and to the 30 kDa antigen, respectively. Conclusions/Significance These findings suggest that the IgG response to Culex salivary gland extracts is suitable as proxy of exposure; however, the specificity to the Culex genus needs further investigation. The lower antigenicity of the 30 kDa recombinant protein represents a limitation to its use. The high specificity of this protein to the Culex genus makes it an attractive candidate and other specific antibody responses might be more relevant as a biomarker of exposure. These epidemiological observations may form a starting point for additional work on developing serological biomarkers of Culex exposure., Author summary The evaluation of exposure to mosquitoes is a key parameter in assessing the risk of transmission of associated pathogens, including zoonoses. Entomological methods represent the gold standard but have several limitations, and efforts are being made to develop new indicators to accurately assess human–Culex contact. This study showed the IgG response to Culex quinquefasciatus salivary gland extract is suitable proxy of exposure to Culex bites. The lower antigenicity of the 30 kDa recombinant protein represents a limitation to its use. The high specificity of this protein to the Culex genus makes it an attractive candidate and other isotypic antibody responses specific to this salivary antigen might be more relevant as a biomarker of exposure.

International audience; Cancer is a major public health issue and represents a significant burden in countries with different levels of economic wealth. In parallel, mosquito-borne infectious diseases represent a growing problem causing significant morbidity and mortality worldwide. Acknowledging that these two concerns are both globally distributed, it is essential to investigate whether they have a reciprocal connection that can fuel their respective burdens. Unfortunately, very few studies have examined the link between these two threats.This review provides an overview of the possible links between mosquitoes, mosquito-borne infectious diseases and cancer. We first focus on the impact of mosquitoes on carcinogenesis in humans including the transmission of oncogenic pathogens through mosquitoes, the immune reactions following mosquito bites, the presence of non-oncogenic mosquito-borne pathogens, and the direct transmission of cancer cells. The second part of this review deals with the direct or indirect consequences of cancer in humans on mosquito behaviour. Thirdly, we discuss the potential impacts that natural cancers in mosquitoes can have on their life history traits and therefore on their vector capacity. Finally, we discuss the most promising research avenues on this topic and the integrative public health strategies that could be envisioned in this context.

Background Malaria is still a major public health concern in Côte d’Ivoire despite mass distribution of long-lasting insecticidal nets (LLINs) as a key preventive strategy. This study intended to evaluate the operational effectiveness of LLINs on the level of human-vector contact using 1 antibody-based biomarker of exposure to Anopheles in urban areas. Methods This cross-sectional study collected socio-demographic data and use of LLINs from 9 neighborhoods in the city of Bouaké (Côte d’Ivoire). Dry blood spots performed in children aged >6 months and adults were used to evaluate immunoglobulin G (IgG) responses to the Anopheles gSG6-P1 salivary peptide. Results IgG response levels to the salivary peptide were significantly lower in individuals who declared having “always” (n = 270) slept under an LLIN compared with those who had “often” (n = 2087) and “never” (n = 88) slept under an LLIN (P Conclusions The human IgG level to this gSG6-P1 salivary peptide could be a useful tool to evaluate the actual effectiveness of LLINs and help design behavioral change interventions that are crucial for sustaining universal coverage.

Background Artemisinin-based combination therapy (ACT), used to treat uncomplicated malaria cases, is one of the main strategies of malaria control and elimination. One of the main objectives of the Benin National Malaria Control Program’s (NMCP) strategic plan is to ensure that at least 80% of uncomplicated malaria is treated with ACT within 24 h. Therefore, it was of great interest to measure whether the country case management of fever amongst children under five, adhered to the NMCP’s strategic plan and look into the barriers to the use of ACT. Methods A cross-sectional survey based on a cluster and multi-stage sampling was conducted in two rural health districts in Benin. We recruited 768 and 594 children under five years were included in the northern and in the southern respectively. Data was collected on the general use of ACT and on the correct use of ACT that adheres to the NMCP’s strategy, as well as the barriers that prevent the proper management of fever amongst children. To assess the certain predictors of ACT usage, logistic regression was used, while taking into account the cluster random effect. Results Among febrile children aged 6 to 59 months, 20.7% in the south and 33.9% in north received ACT. The correct use of ACT, was very low, 5.8% and in southern and 8.6% northern areas. Caregivers who received information on ACT were 3.13 time more likely in the south and 2.98 time more likely in the north to give ACT to their feverish child, PPR = 3.13[1.72–4.15] and PPR = 2.98 [2.72–3.11] respectively. Chloroquine and quinine, other malaria treatments not recommended by NMCP, were still being used in both areas: 12.3 and 3.3% in the south and 11.4 and 3.0% in the north. Conclusion In Benin, the use and the correct use of ACT for febrile children remains low. The study also showed that having received information about the use of ACT is positively associated with the use of ACT. This point highlights the fact that efforts may not have been sufficiently integrated with social communication, which should be based on the behavioural determinants of populations. Electronic supplementary material The online version of this article (10.1186/s12889-018-5077-6) contains supplementary material, which is available to authorized users.

Background In malaria-endemic areas, human populations are frequently exposed to immunomodulatory salivary components injected during mosquito blood feeding. The consequences on pathogen-specific immune responses are not well known. This study evaluated and compared the humoral responses specific to merozoite stage vaccine candidates of Plasmodium falciparum, in children differentially exposed to Anopheles bites in a natural setting. Methods The cross-sectional study was carried out in Bouaké (Côte d’Ivoire) where entomological data and blood samples from children (0–14 years) were collected in two sites with similar malaria prevalence. Antibody (IgG, IgG1, IgG3) responses to PfAMA1 and PfMSP1 were evaluated by ELISA. Univariate and multivariate analysis were performed to assess the relationship between the immune responses to P. falciparum antigens and exposure to Anopheles bites in the total cohort and in each site, separately. The individual level of exposure to Anopheles bites was evaluated by quantifying specific IgG response to the Anopheles gSG6-P1 salivary peptide, which represents a proxy of Anopheles exposure. Results The anti-Plasmodium humoral responses were different according to the level of exposure of children, with those highly exposed to Anopheles presenting significantly lower antibody responses to PfMSP1 in total population (IgG and IgG3) and in Petessou village (IgG, IgG1, IgG3). No significant difference was seen for PfAMA1 antigen between children differently exposed to Anopheles. In Dar-es-Salam, a neighbourhood where a high Culex density was reported, children presented very low antibody levels specific to both antigens, and no difference according to the exposure to Anopheles bites was found. Conclusion These findings may suggest that immunomodulatory components of Anopheles saliva, in addition to other factors, may participate to the modulation of the humoral response specific to Plasmodium merozoite stage antigens. This epidemiological observation may form a starting point for additional work to decipher the role of mosquito saliva on the modulation of the anti-Plasmodium acquired immunity and clinical protection in combining both field and ex vivo immunological studies.

Background Anarchic and poorly controlled urbanization led to an increased risk of mosquito-borne diseases (MBD) in many African cities. Here, we evaluate the spatial heterogeneity of human exposure to malaria and arboviral disease vectors in an urban area of northern Senegal, using antibody-based biomarkers of exposure to Anopheles and Aedes mosquito bites. Methods A cross-sectional study was undertaken during the rainy season of 2014 in 4 neighborhoods of Saint-Louis, a city in northern Senegal. Among children aged 6–59 months in each neighborhood, the dried blood spot technique was used to evaluate immunoglobulin G (IgG) responses to both gSG6-P1 (Anopheles) and Nterm–34-kDa (Aedes) salivary peptides as validated biomarkers of respective mosquito bite exposure. Results IgG response levels to gSG6-P1 and Nterm–34-kDa salivary peptides varied significantly between the 4 neighborhoods (P < .0001). The level of exposure to Aedes bites also varied according to household access to sanitation services (P = .027), whereas that of exposure to Anopheles bites varied according to insecticide-treated bed net use (P = .006). In addition, spatial clusters of high contact between humans and mosquitoes were identified inside 3 neighborhoods. Conclusions Antibody-based biomarkers of exposure to Anopheles and Aedes mosquito bites could be helpful tools for evaluating the heterogeneity of exposure to malaria and arboviral disease vectors by national control programs.

International audience; Urban malaria is an underestimated serious health concern in African countries. This study aimed to evaluate the risk of malaria transmission in an urban area by evaluating the level of human exposure to Anopheles bites using an Anopheles salivary biomarker (gambiae Salivary Gland Protein-6 peptide 1 [gSG6-P1] peptide). Two multidisciplinary cross-sectional studies were undertaken in five sites of Bouaké city (three urban districts and two surrounding villages, used as control; Côte d’Ivoire) during the rainy season and the dry season. Blood samples were obtained from children 6 months to 14 years of age for immunological tests. The level of anti-gSG6-P1 immunoglobulin G (IgG) antibodies was significantly higher in the rainy season than the dry season in both urban and rural sites (P < 0.0001). Interestingly, children with the highest anti-gSG6-P1 IgG responses in the rainy season were infected by Plasmodium falciparum. Surprisingly, no difference of anti-gSG6-P1 IgG level was observed between urban and rural areas, for either season. The current data suggest that children in the urban city of Bouaké could be as highly exposed to Anopheles bites as children living in surrounding villages. The immunological biomarker of human exposure to Anopheles bites may be used to accurately assess the potential risk of malaria transmission in African urban settings.

The study of human–vector immune relationships could provide several avenues for the control of vector-borne diseases such as malaria. This chapter describes the epidemiological relationships of antisaliva antibody responses and blood-feeding arthropod density that gave birth to the concept of salivary biomarkers of human exposure to Anopheles bites. Approaches, techniques, and methods used to develop and validate specific candidate-biomarkers of exposure to Anopheles mosquito bites are highlighted with a focus on their potential applications in malaria-endemic areas. Development of this salivary biomarker into a rapid diagnostic test, such as a malaria rapid diagnostic test, will enable rapid and easy detection in finger prick blood of human antibodies to Anopheles gSG6-P1 salivary peptide. Such an assay will allow operational research (identification of populations at risk of malaria, survey of area for malaria elimination, evaluation of vector control strategies) by national malaria control programs for better control of malaria.

In the context of the Pan African Tsetse and Trypanosomiasis Eradication Campaign, the value of tsetse saliva antibodies as a biomarker of cattle exposure to tsetse flies was evaluated, as this could provide an alternative and complementary tool to conventional entomological methods. Serum immune reactivity to Glossina (G.) palpalis (p.)gambiensis, G. tachinoides and G. morsitans (m.) submorsitans whole saliva extracts (WSE) were monitored in cattle from both tsetse free and tsetse infested areas, and in cows experimentally exposed to tsetse flies and other hematophagous arthropods. In the tsetse infested area, cattle IgG responses to Glossina WSE were significantly higher during the dry season (p < 0.0001) when herds are most exposed to tsetse flies and in infected animals (p = 0.01) as expected in the case of a biomarker of exposure. Experimental studies further confirmed this as a quick rise of specific IgGs was observed in animals exposed to tsetse flies (within weeks), followed by a rapid clearance after exposure was stopped. In contrast to the two other tsetse species, G. m. submorsitans WSE enabled to detect exposure to all tsetse species and were associated with low level of cross-reactivity to other blood sucking arthropods. Finally, IgG responses to G. m. submorsitans salivary antigens enabled to distinguish different groups of cows according to exposure levels, thus indicating that tsetse saliva antibodies are not only indicators of tsetse exposure but also are correlated to the intensity of tsetse contacts (p = 0.0031). Implementation of this new sero-epidemiological marker of cattle exposure to tsetse flies in the framework of tsetse elimination campaigns is discussed.

International audience; BACKGROUND:In a context of large-scale implementation of malaria vector control tools, such as the distribution of long-lasting insecticide nets (LLIN), it is necessary to regularly assess whether strategies are progressing as expected and then evaluate their effectiveness. The present study used the case-control approach to evaluate the effectiveness of LLIN 42 months after national wide distribution. This study design offers an alternative to cohort study and randomized control trial as it permits to avoid many ethical issues inherent to them.METHODS:From April to August 2011, a case-control study was conducted in two health districts in Benin; Ouidah-Kpomasse-Tori (OKT) in the south and Djougou-Copargo-Ouake (DCO) in the north. Children aged 0-60 months randomly selected from community were included. Cases were children with a high axillary temperature (≥37.5 °C) or a reported history of fever during the last 48 h with a positive rapid diagnostic test (RDT). Controls were children with neither fever nor signs suggesting malaria with a negative RDT. The necessary sample size was at least 396 cases and 1188 controls from each site. The main exposure variable was "sleeping every night under an LLIN for the 2 weeks before the survey" (SL). The protective effectiveness (PE) of LLIN was calculated as PE = 1 - odds ratio.RESULTS:The declared SL range was low, with 17.0 and 27.5 % in cases and controls in the OKT area, and 44.9 and 56.5 % in cases and controls, in the DCO area, respectively. The declared SL conferred 40.5 % (95 % CI 22.2-54.5 %) and 55.5 % (95 % CI 28.2-72.4 %) protection against uncomplicated malaria in the OKT and the DCO areas, respectively. Significant differences in PE were observed according to the mother's education level.CONCLUSION:In the context of a mass distribution of LLIN, their use still conferred protection in up to 55 % against the occurrence of clinical malaria cases in children. Social factors, the poor use and the poor condition of an LLIN can be in disfavour with its effectiveness. In areas, where LLIN coverage is assumed to be universal or targeted at high-risk populations, case-control studies should be regularly conducted to monitor the effectiveness of LLIN. The findings will help National Malaria Control Programme and their partners to improve the quality of malaria control according to the particularity of each area or region as far as possible

International audience; The widespread implementation of long-lasting insecticidal nets (LLINs) is a major intervention method for malaria control. Although the LLINs coverage increases, information available on the physical integrity (PI) of implemented LLINs is incomplete. This study aimed to validate human IgG antibody (Ab) response to Anopheles gSG6-P1 salivary peptide antigen, previously demonstrated as a pertinent biomarker of human exposure to Anopheles bites, for evaluating the PI of LLINs in field conditions. We analyzed data from 262 randomly selected children (< 5 years of age) in health districts of Benin. Anti-gSG6-P1 IgG responses were assessed and compared with the PI of LLINs that these same children slept under, and evaluated by the hole index (HI). Specific IgG levels were positively correlated to LLINs HI (r = 0.342; P < 0.0001). According to antipeptide IgG level (i.e., intensity of vector exposure), two categories of LLINs PI were defined: 1) group “HI: [0, 100]” corresponding to LLINs with “good” PI and 2) “HI > 100” corresponding to LLINs with “bad” PI. These results suggest that human Ab response to salivary peptide could be a complementary tool to help defining a standardized threshold of efficacy for LLINs under field use.

Background: During blood meal, the female mosquito injects saliva able to elicit an immune response in the vertebrate. This immune response has been proven to reflect the intensity of exposure to mosquito bites and risk of infection for vector transmitted pathogens such as malaria. The peptide gSG6-P1 of An. gambiae saliva has been demonstrated to be antigenic and highly specific to Anopheles as a genus. However, the applicability of gSG6-P1 to measure exposure to different Anopheles species endemic in the Americas has yet to be evaluated. The purpose of this pilot study was to test whether human participants living in American countries present antibodies able to recognize the gSG6-P1, and whether these antibodies are useful as a proxy for mosquito bite exposure and malaria risk. Methods: We tested human serum samples from Colombia, Chile, and the United States for the presence of IgG antibodies against gSG6-P1 by ELISA. Antibody concentrations were expressed as delta optical density (Delta OD) of each sera tested in duplicates. The difference in the antibody concentrations between groups was tested using the nonparametric Mann Whitney test (independent groups) and the nonparametric Wilcoxon matched-pairs signed rank test (dependent groups). All differences were considered significant with a P < 0.05. Results: We found that the concentration of gSG6-P1 antibodies was significantly correlated with malaria infection status and mosquito bite exposure history. People with clinical malaria presented significantly higher concentrations of IgG anti-gSG6-P1 antibodies than healthy controls. Additionally, a significant raise in antibody concentrations was observed in subjects returning from malaria endemic areas. Conclusion: Our data shows that gSG6-P1 is a suitable candidate for the evaluation of exposure to Anopheles mosquito bites, risk of malaria transmission, and effectiveness of protection measures against mosquito bites in the Americas.

International audience; Background: The estimates of risk of malaria in early childhood are imprecise given the current entomologic and parasitological tools. Thus, the utility of anti-Anopheles salivary gSG6-P1 peptide antibody responses in measuring exposure to Anopheles bites during early infancy has been assessed.Methods: Anti-gSG6-P1 IgG and IgM levels were evaluated in 133 infants (in Benin) at three (M3), six (M6), nine (M9) and 12 (M12) months of age. Specific IgG levels were also assessed in their respective umbilical cord blood (IUCB) and maternal blood (MPB).Results: At M3, 93.98 and 41.35% of infants had anti-gSG6-P1 IgG and IgM Ab, respectively. Specific median IgG and IgM levels gradually increased between M3 and M6 (p < 0.0001 and p < 0.001), M6–M9 (p < 0.0001 and p = 0.085) and M9–M12 (p = 0.002 and p = 0.03). These levels were positively associated with the Plasmodium falciparum infection intensity (p = 0.006 and 0.003), and inversely with the use of insecticide-treated bed nets (p = 0.003 and 0.3). Levels of specific IgG in the MPB were positively correlated to those in the IUCB (R = 0.73; p < 0.0001) and those at M3 (R = 0.34; p < 0.0001).Conclusion: The exposure level to Anopheles bites, and then the risk of malaria infection, can be evaluated in young infants by assessing anti-gSG6-P1 IgM and IgG responses before and after 6-months of age, respectively. This tool can be useful in epidemiological evaluation and surveillance of malaria risk during the first year of life.

Aedes mosquitoes are the major vectors of (re)-emerging infections including arboviruses (dengue, Chikungunya, yellow fever) in developing countries. Moreover, the emergence of Aedes-borne diseases in the developed world is currently a source of concern. Evaluation of human immune responses to Aedes bites could be a useful immuno-epidemiological tool for evaluating exposure to Aedes-borne diseases and thus predicting the risk of such emerging diseases. Specific IgE and IgG4 antibody (Ab) responses to Aedes aegypti saliva were evaluated in young Senegalese children living in an area of exposure to the Aedes vector. Specific IgE and IgG4 responses increased during rainy season of high exposure to Aedes bites. In addition, the evolution of anti-saliva isotype levels during the rainy season presented spatial heterogeneity between the studied villages. These preliminaries results support the potential approach of using anti-saliva Ab responses for evaluating exposure to Aedes vectors and risks of emerging arbovirus infections.

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. Electronic supplementary material The online version of this article (doi:10.1186/s13071-014-0599-y) contains supplementary material, which is available to authorized users.

Malaria and schistosomiasis are the two major parasite diseases present in developing countries. The epidemiological co-infection with schistosomiasis could influence the development of the physiological reaction associated with Plasmodium falciparum infection in human. Most studies have demonstrated the association of circulating levels of interferon-gamma (IFN-gamma), tumour necrosis factor-a (TNF-alpha), interleukin-10 (IL-10), transforming growth factor (TGF-beta) and soluble Tumour Necrosis Factor Receptors (sTNF-RI and sTNF-RII) with the morbidity of malaria. In the present study, we showed that Schistosoma haematobium co-infection influences, in an age-dependent manner, the unbalance between pro- and anti-inflammatory circulating cytokines that play a key role during malaria infection. Indeed, children co-infected by S. haematobium have higher levels of IFN-gamma and sTNF-RII than children infected only by P. falciparum. In contrast, co-infected adults presented a significant increase of IFN-gamma, IL-10, TGF-beta, sTNF-RI and sTNF-RII rates and IL-10/TNF-alpha ratio. Taken together, this study indicates that schistosomiasis co-infection can unbalance the regulation of inflammatory factors in uncomplicated P. falciparum malaria. The possible consequences of the schistosomiasis co-infection for age-dependent malaria morbidity are discussed.

The epidemiological coexistence of schistosomiasis and malaria is frequently observed in developing countries. Co-infection with malaria in children could influence the development of acquired immunity associated with the resistance or the pathology of schistosomiasis. In the present study, performed during May to June 1996 in Senegal, the humoral immune response to Schistosoma haematobium 28 kDa glutathione S-transferase (Sh28GST) vaccinal antigen and to soluble egg antigens (SEA) has been evaluated in individuals infected by S. haematobium. Specific immunoglobulin G3 (IgG3) and IgE responses were significantly higher in co-infected children with Plasmodium falciparum compared with children infected with S. haematobium only. In addition, circulating levels of interferon-gamma (IFN-gamma), interleukin-10 (IL-10), and soluble tumor necrosis factor receptor II (sTNF-RII), 3 parameters associated with schistosomiasis morbidity, were significantly increased in co-infected children. Taken together, this study indicated that malaria co-infection can both influence the acquired specific immune response to schistosome antigens and unbalance the regulation of inflammatory factors closely involved in schistosomiasis pathology.

Vector control is one of the most valuable strategies to fight the expanding distribution of arthropod-borne diseases (ABDs). However, its implementation and monitoring are time consuming and labor intensive that may impede regular entomological survey in the field. To increase the impact of vector control strategies in fighting ABDs, the current entomological methods should be improved by new complementary tools. The exploration of the close interactions between the human host and the vector through the antigenic salivary proteins of hematophagous arthropods has lead to the development of new salivary biomarkers (SBs). This chapter focuses on the methodologies used to develop the SB of vector bites. The relevancy of these immunological biomarkers in assessing the human exposure to vector bites and their capacity to evaluate the efficacy of vector control strategies as well as the risk of pathogen transmission are also discussed. List of Abbreviations Ab Antibody ABDs Arthropod-Borne Diseases ADP Adenosine Diphosphate Ae. Aedes An. Anopheles gSG6 Anopheles gambiae Salivary Protein Ig Immunoglobulin ITN Insecticide-Treated Net kDa Kilodalton Lu. Lutzomyia P. Phlebotomus SB Salivary Biomarker SGEs Salivary Gland Extracts Th T Helper VCP Vector Control Program 1178 S. Doucoure et al.

Background: One of the control tools to reduce malaria transmission is the use of LLINs. However, several studies show that household bed net use is quite low. A study was developed to better understand the cultural factors that might explain these gaps in Benin. One reason mentioned is that bed nets can catch on fire and cause harm. This paper presents a summary of these findings, their analysis and the ensuing issues. Methods: This anthropological study is based on an inductive qualitative approach, including 91 semi-structured interviews conducted from July 2011 to March 2012 in a health district in Southern Benin. Results: Fifty-six persons stated that bed nets can catch on fire but do not always refer to specific facts. However, 34 of the 56 people narrate specific events they heard or experienced. 39 accounts were geographically located and situated in time, with various details. In 27 situations, people were burned, for which 12 people reportedly died. Discussion: The disparity between these results and the dearth of bibliographic documentation in the initial search prompted a more in-depth literature review: 16 contributions between 1994 and 2013 were found. Bed net fires were noted in 10 countries, but it is impossible to ascertain the frequency of such events. Moreover, bodily harm can be significant, and several cases of death attributed to bed net fires were noted. Conclusions: Indisputably, the use of bed nets to reduce the impact of this terrible disease is an optimal control method. However, the perception that LLINs have a potentially negative effect hinders the use rate in the real world, at least for some. If some people fear the risk of fires, this possibility must be addressed during information and prevention sessions on malaria, with a communication strategy tailored to specific social contexts. Moreover, all possible measures should be taken to limit the harm suffered by individuals and their families.

International audience; Insensitive acetylcholinesterase resistance due to a mutation in the acetylcholinesterase (ace) encoding ace-1 gene confers cross-resistance to organophosphate and carbamate insecticides in Anopheles gambiae populations from Central and West Africa. This mutation is associated with a strong genetic cost revealed through alterations of some life history traits but little is known about the physiological and behavioural changes in insects bearing the ace-1(R) allele. Comparative analysis of the salivary gland contents between An. gambiae susceptible and ace-1(R) resistant strains was carried out to charaterize factors that could be involved in modifications of blood meal process, trophic behaviour or pathogen interaction in the insecticide-resistant mosquitoes. Differential analysis of the salivary gland protein profiles revealed differences in abundance for several proteins, two of them showing major differences between the two strains. These two proteins identified as saglin and TRIO are salivary gland-1 related proteins, a family unique to anopheline mosquitoes, one of them playing a crucial role in salivary gland invasion by Plasmodium falciparum sporozoites. Differential expression of two other proteins previously identified in the Anopheles sialome was also observed. The differentially regulated proteins are involved in pathogen invasion, blood feeding process, and protection against oxidation, relevant steps in the outcome of malaria infection. Further functional studies and insect behaviour experiments would confirm the impact of the modification of the sialome composition on blood feeding and pathogen transmission abilities of the resistant mosquitoes. The data supports the hypothesis of alterations linked to insecticide resistance in the biology of the primary vector of human malaria in Africa.

Author(s): Ya-Umphan, Phubeth; Cerqueira, Dominique; Parker, Daniel M; Cottrell, Gilles; Poinsignon, Anne; Remoue, Franck; Brengues, Cecile; Chareonviriyaphap, Theeraphap; Nosten, Francois; Corbel, Vincent | Abstract: BackgroundThe modalities of malaria transmission along the Thailand-Myanmar border are poorly understood. Here we address the relevance of using a specific Anopheles salivary biomarker to measure the risk among humans of exposure to Anopheles bites.MethodsSerologic surveys were conducted from May 2013 to December 2014 in 4 sentinel villages. More than 9400 blood specimens were collected in filter papers from all inhabitants at baseline and then every 3 months thereafter, for up to 18 months, for analysis by enzyme-linked immunosorbent assay. The relationship between the intensity of the human antibody response and entomological indicators of transmission (human biting rates and entomological inoculation rates [EIRs]) was studied using a multivariate 3-level mixed model analysis. Heat maps for human immunoglobulin G (IgG) responses for each village and survey time point were created using QGIS 2.4.ResultsThe levels of IgG response among participants varied significantly according to village, season, and age (Pl.001) and were positively associated with the abundance of total Anopheles species and primary malaria vectors and the EIR (Pl.001). Spatial clusters of high-IgG responders were identified across space and time within study villages.ConclusionsThe gSG6-P1 biomarker has great potential to address the risk of transmission along the Thailand-Myanmar border and represents a promising tool to guide malaria interventions.

Background: Evaluation of malaria sporozoite rates in the salivary glands of Anopheles gambiae is essential for estimating the number of infective mosquitoes, and consequently, the entomological inoculation rate (EIR). EIR is a key indicator for evaluating the risk of malaria transmission. Although the enzyme-linked immunosorbent assay specific for detecting the circumsporozoite protein (CSP-ELISA) is routinely used in the field, it presents several limitations. A multiplex PCR can also be used to detect the four species of Plasmodium in salivary glands. The aim of this study was to evaluate the efficacy of a real-time quantitative PCR in detecting and quantifying wild Plasmodium falciparum in the salivary glands of An. gambiae. Methods: Anopheles gambiae (n=364) were experimentally infected with blood from P. falciparum gametocyte carriers, and P. falciparum in the sporozoite stage were detected in salivary glands by using a real-time quantitative PCR (qPCR) assay. The sensitivity and specificity of this qPCR were compared with the multiplex PCR applied from the Padley method. CSP-ELISA was also performed on carcasses of the same mosquitoes. Results: The prevalence of P. falciparum and the intensity of infection were evaluated using qPCR. This method had a limit of detection of six sporozoites per mu L based on standard curves. The number of P. falciparum genomes in the salivary gland samples reached 9,262 parasites/mu L (mean: 254.5; 95% CI: 163.5-345.6). The qPCR showed a similar sensitivity (100%) and a high specificity (60%) compared to the multiplex PCR. The agreement between the two methods was "substantial" (k = 0.63, P < 0.05). The number of P. falciparum-positive mosquitoes evaluated with the qPCR (76%), multiplex PCR (59%), and CSP-ELISA (83%) was significantly different (P < 0.005). Conclusions: The qPCR assay can be used to detect P. falciparum in salivary glands of An. gambiae. The qPCR is highly sensitive and is more specific than multiplex PCR, allowing an accurate measure of infective An. gambiae. The results also showed that the CSP-ELISA overestimates the sporozoite rate, detecting sporozoites in the haemolymph in addition to the salivary glands.

The saliva of blood sucking arthropods contains a number of pharmacologically active compounds that induce an antibody response in exposed human individuals. The objectives of the present study were (i) to assess the human IgG response directed against salivary antigens of Glossina palpalis gambiensis, the main vector of Trypanosoma brucei gambiense in West Africa, as a biomarker of human tsetse contacts; and (ii) to identify specific salivary antigens. Immune reactivity of human plasma collected within active human African trypanosomiasis (HAT) foci (coastal Guinea), historical foci where tsetse flies are still present (South-West Burkina Faso) and a tsetse free area (Bobo-Dioulasso, Burkina Faso), was measured by ELISA against whole saliva extracts. In the active HAT foci and areas where tsetse flies were present in high densities, specific IgG responses were significantly higher (p < 0.0001) to those in Bobo-Dioulasso or in Loropeni, where tsetse flies were either absent or only present at low densities. Furthermore, 2D-electrophoresis combined with mass spectrometry enabled to reveal that several antigens were specifically recognized by plasma from exposed individuals. Among them, four salivary proteins were successfully identified (Ada, 5'Nuc, Ag5 and Tsgf1). These results represent a first attempt to identify Glossina salivary proteins or synthetic peptides to develop a standardized and specific biomarker of tsetse exposure in West Africa.

International audience; BACKGROUND: Over the past decade, a sharp decline of malaria burden has been observed in several countries. Consequently, the conventional entomological methods have become insufficiently sensitive and probably under-estimate micro-geographical heterogeneity of exposure and subsequent risk of malaria transmission. In this study, we investigated whether the human antibody (Ab) response to Anopheles salivary gSG6-P1 peptide, known as a biomarker of Anopheles exposure, could be a sensitive and reliable tool for discriminating human exposure to Anopheles bites in area of low and seasonal malaria transmission. METHODS: A multi-disciplinary survey was performed in Northern Senegal where An. gambiae s.l. is the main malaria vector. Human IgG Ab response to gSG6-P1 salivary peptide was compared according to the season and villages in children from five villages in the middle Senegal River valley, known as a low malaria transmission area. RESULTS: IgG levels to gSG6-P1 varied considerably according to the villages, discriminating the heterogeneity of Anopheles exposure between villages. Significant increase of IgG levels to gSG6-P1 was observed during the peak of exposure to Anopheles bites, and decreased immediately after the end of the exposure season. In addition, differences in the season-dependent specific IgG levels between villages were observed after the implementation of Long-Lasting Insecticidal Nets by The National Malaria Control Program in this area. CONCLUSION: The gSG6-P1 salivary peptide seems to be a reliable tool to discriminate the micro-geographical heterogeneity of human exposure to Anopheles bites in areas of very low and seasonal malaria transmission. A biomarker such as this could also be used to monitor and evaluate the possible heterogeneous effectiveness of operational vector control programs in low-exposure areas.

Aim: In Benin, artemisinin-based combination therapy (ACT) has been recommended as the first-line treatment for uncomplicated Plasmodium falciparum malaria since 2004. The emergence in Southeast Asia of parasites that are resistant to artemisinins poses a serious threat to global control of this disease. The presence of artemisinin resistance genotypes in parasite populations in Benin is currently unknown. The present study investigated the prevalence of relevant K13-propeller gene polymorphisms in parasite isolates from the north-western region of Benin. Method: Plasmodium falciparum isolates were collected from children with a confirmed diagnosis of malaria aged 6 months to 5 years in two towns, Cobly and Djougou, in the north-western part of Benin. The study was conducted during the rainy season from July to November 2014 in local health facilities. The K13-propeller gene was amplified in parasite isolates using nested PCR and subsequently sequenced. Results: A total of 108 children were recruited into the study. The efficiency of amplification reactions was 72% (78/108). The propeller domain of the K13 gene was successfully sequenced in 78 P. falciparum isolates; all of them were wild type with no polymorphisms detectable. Conclusion: The absence of mutations in the K13 gene indicates that P. falciparum parasite populations in the study area are still fully susceptible to artemisinins.

Background The existing metrics of malaria transmission are limited in sensitivity under low transmission intensity. Robust surveillance systems are needed as interventions to monitor reduced transmission and prevention of rapid reintroduction. Serological tools based on antibody responses to parasite and vector antigens are potential tools for transmission measurements. The current study sought to evaluate antibody responses to Anopheles gambiae salivary gland peptide (gSG6- P1), as a biomarker of human exposure to Anopheles bites, in different transmission settings and seasons. The comparison between anti-MSP-119 IgG immune responders and non-responders allowed exploring the robustness of the gSG6-P1 peptide as a surveillance tool in an area of decreasing malaria transmission. Methods Total IgG levels to gSG6-P1 were measured in an age-stratified cohort (< 5, 5–14 and ≥ 15 years) in a total of 1,366 participants from three localities in western Kenya [Kisii (hypoendemic), Kakamega (mesoendemic), and Kombewa (hyperendemic)] including 607 sera that were additionally tested for MSP-119 specific responses during a low and a high malaria transmission seasons. Antibody prevalence and levels were compared between localities with different transmission intensities. Regression analysis was performed to examine the association between gSG6-P1 and MSP-119 seroprevalence and parasite prevalence. Result Seroprevalence of gSG6-P1 in the uphill population was 36% while it was 50% valley bottom (χ2 = 13.2, df = 1, p

International audience; Standard entomological methods for evaluating the impact of vector control lack sensitivity in low-malaria-risk areas. The detection of human IgG specific to Anopheles gSG6-P1 salivary antigen reflects a direct measure of human-vector contact. This study aimed to assess the effectiveness of a range of vector control measures (VCMs) in urban settings by using this biomarker approach. The study was conducted from October to December 2008 on 2,774 residents of 45 districts of urban Dakar. IgG responses to gSG6-P1 and the use of malaria VCMs highly varied between districts. At the district level, specific IgG levels significantly increased with age and decreased with season and with VCM use. The use of insecticide-treated nets, by drastically reducing specific IgG levels, was by far the most efficient VCM regardless of age, season or exposure level to mosquito bites. The use of spray bombs was also associated with a significant reduction of specific IgG levels, whereas the use of mosquito coils or electric fans/air conditioning did not show a significant effect. Human IgG response to gSG6-P1 as biomarker of vector exposure represents a reliable alternative for accurately assessing the effectiveness of malaria VCM in low-malaria-risk areas. This biomarker tool could be especially relevant for malaria control monitoring and surveillance programmes in low-exposure/low-transmission settings.

Background Malaria transmission occurs during the blood feeding of infected anopheline mosquitoes concomitant with a saliva injection into the vertebrate host. In sub-Saharan Africa, most malaria transmission is due to Anopheles funestus s.s and to Anopheles gambiae s.l. (mainly Anopheles gambiae s.s. and Anopheles arabiensis). Several studies have demonstrated that the immune response against salivary antigens could be used to evaluate individual exposure to mosquito bites. The aim of this study was to assess the use of secreted salivary proteins as specific biomarkers of exposure to An. gambiae and/or An. funestus bites. Methods For this purpose, salivary gland proteins 6 (SG6) and 5′nucleotidases (5′nuc) from An. gambiae (gSG6 and g-5′nuc) and An. funestus (fSG6 and f-5′nuc) were selected and produced in recombinant form. The specificity of the IgG response against these salivary proteins was tested using an ELISA with sera from individuals living in three Senegalese villages (NDiop, n = 50; Dielmo, n = 38; and Diama, n = 46) that had been exposed to distinct densities and proportions of the Anopheles species. Individuals who had not been exposed to these tropical mosquitoes were used as controls (Marseille, n = 45). Results The IgG responses against SG6 recombinant proteins from these two Anopheles species and against g-5′nucleotidase from An. gambiae, were significantly higher in Senegalese individuals compared with controls who were not exposed to specific Anopheles species. Conversely, an association was observed between the level of An. funestus exposure and the serological immune response levels against the f-5′nucleotidase protein. Conclusion This study revealed an Anopheles salivary antigenic protein that could be considered to be a promising antigenic marker to distinguish malaria vector exposure at the species level. The epidemiological interest of such species-specific antigenic markers is discussed.

Background Urban malaria can be a serious public health problem in Africa. Human-landing catches of mosquitoes, a standard entomological method to assess human exposure to malaria vector bites, can lack sensitivity in areas where exposure is low. A simple and highly sensitive tool could be a complementary indicator for evaluating malaria exposure in such epidemiological contexts. The human antibody response to the specific Anopheles gSG6-P1 salivary peptide have been described as an adequate tool biomarker for a reliable assessment of human exposure level to Anopheles bites. The aim of this study was to use this biomarker to evaluate the human exposure to Anopheles mosquito bites in urban settings of Dakar (Senegal), one of the largest cities in West Africa, where Anopheles biting rates and malaria transmission are supposed to be low. Methods One cross-sectional study concerning 1,010 (505 households) children (n = 505) and adults (n = 505) living in 16 districts of downtown Dakar and its suburbs was performed from October to December 2008. The IgG responses to gSG6-P1 peptide have been assessed and compared to entomological data obtained in or near the same district. Results Considerable individual variations in anti-gSG6-P1 IgG levels were observed between and within districts. In spite of this individual heterogeneity, the median level of specific IgG and the percentage of immune responders differed significantly between districts. A positive and significant association was observed between the exposure levels to Anopheles gambiae bites, estimated by classical entomological methods, and the median IgG levels or the percentage of immune responders measuring the contact between human populations and Anopheles mosquitoes. Interestingly, immunological parameters seemed to better discriminate the exposure level to Anopheles bites between different exposure groups of districts. Conclusions Specific human IgG responses to gSG6-P1 peptide biomarker represent, at the population and individual levels, a credible new alternative tool to assess accurately the heterogeneity of exposure level to Anopheles bites and malaria risk in low urban transmission areas. The development of such biomarker tool would be particularly relevant for mapping and monitoring malaria risk and for measuring the efficiency of vector control strategies in these specific settings.

International audience; Human antibody (Ab) response to Anopheles whole saliva, used as biomarker of Anopheles exposure, was investigated over a period of two years (2008–2009), in children between 2 to 9 years old, before and after the introduction of three different malaria vector control methods; deltamethrin treated long lasting impregnated nets (LLIN) and insecticide treated plastic sheeting (ITPS) - Zero Fly®) (ITPS-ZF), deltamethrin impregnated Durable (Wall) Lining (ITPS-DL – Zerovector®) alone, and indoor residual spraying (IRS) with lambdacyhalothrin alone. These different vector control methods resulted in considerable decreases in all three entomological (82.4%), parasitological (54.8%) and immunological criteria analyzed. The highest reductions in the number of Anopheles collected and number of positive blood smears, respectively 82.1% and 58.3%, were found in Capango and Canjala where LLIN and ITPS-ZF were implemented. The immunological data based on the level of anti-saliva IgG Ab in children of all villages dropped significantly from 2008 to 2009, except in Chissequele. These results indicated that these three vector control methods significantly reduced malaria infections amongst the children studied and IRS significantly reduced the human-Anopheles contact. The number of Anopheles, positive blood smears, and the levels of anti-saliva IgG Ab were most reduced when LLIN and ITPS-ZF were used in combination, compared to the use of one vector control method alone, either ITPS-DL or IRS. Therefore, as a combination of two vector control methods is significantly more effective than one control method only, this control strategy should be further developed at a more global scale.

a b s t r a c t Malaria immunity is modulated by many environmental and epidemiological factors. This study evaluates the influence of a hitherto unstudied environmental-epidemiological fac- tor, namely the impact of human exposure to Anopheles bites on the isotype profile of acquired antibody responses to Plasmodium falciparum. In two Senegalese villages where the intensity of exposure to Anopheles bites was markedly different (high and low exposure), specific IgG1 and IgG3 responses to P. falciparum whole schizont extract (WSE) and circum- sporozoite protein (CSP) were evaluated at the peak of Anopheles exposure (September) and later (December) in a cohort of 120 children aged 3-8 years. Multivariate analysis showed a significantly lower IgG1 response against P. falciparum WSE and CSP in children highly exposed to Anopheles bites (Gankette) compared to those who were weakly exposed (Mboula). In contrast, in both villages, parasitemia and increasing age were strongly asso- ciated with higher IgG1 and IgG3 levels. We hypothesize that high exposure to Anopheles bites could inhibit IgG1-dependent responsiveness to P. falciparum known to induce protec- tive immune responses against malaria. The impact of mosquito saliva on the regulation of specific protective immunity may need to be taken into account in epidemiological studies and trials for malaria vaccines.

Background The evaluation of malaria transmission intensity is a crucial indicator for estimating the burden of malarial disease. In this respect, entomological and parasitological methods present limitations, especially in low transmission areas. The present study used a sensitive multiplex assay to assess the exposure to Plasmodium falciparum infection in children living in an area of low endemicity. In three Senegalese villages, specific antibody (IgG) responses to 13 pre-erythrocytic P. falciparum peptides derived from Lsa1, Lsa3, Glurp, Salsa, Trap, Starp, Csp and Pf11.1 proteins were simultaneously evaluated before (June), at the peak (September) and after (December) the period of malaria transmission, in children aged from 1 to 8 years. Results Compared to other antigens, a high percentage of seropositivity and specific antibody levels were detected with Glurp, Salsa1, Lsa3NR2, and Lsa1J antigens. The seropositivity increased with age for all tested antigens. Specific IgG levels to Glurp, Salsa1, Lsa3NR2, and Lsa1J were significantly higher in P. falciparum infected children compared to non-infected and this increase is significantly correlated with parasite density. Conclusion The multiplex assay represents a useful technology for a serological assessment of rapid variations in malaria transmission intensity, especially in a context of low parasite rates. The use of such combined serological markers (i.e. Glurp, Lsa1, Lsa3, and Salsa) could offer the opportunity to examine these variations over time, and to evaluate the efficacy of integrated malaria control strategies.

Although domestic animals may not be permissive for Plasmodium, they could nevertheless play a role in the epidemiology of malaria by attracting Anopheles away from humans. To investigate interactions between domestic animals and mosquitoes, we assayed immunoglobulin G (IgG) antibodies directed against the salivary proteins of Anopheles gambiae in domestic animals living in Senegalese villages where malaria is endemic. By Western blotting, sera from bovines (n = 6), ovines (n = 36), and caprines (n = 36) did not react with Anopheles whole saliva. In contrast, equine sera recognized proteins in both saliva and salivary gland extracts. Two of the major immunogens (32 and 72 kDa) were also reactive in extracts from other major mosquito genera (Aedes and Culex), but reactions to Anopheles-specific antigens were detected in 12 of 17 horses. These data suggest that horses strongly react to Anopheles bites, and further experiments on horses are warranted to investigate the impact of this domestic animal species on the transmission of human malaria.

Salivary proteins injected by blood feeding arthropods into their hosts evoke a saliva-specific humoral response which can be useful to evaluate exposure to bites of disease vectors. However, saliva of hematophagous arthropods is a complex cocktail of bioactive factors and its use in immunoassays can be misleading because of potential cross-reactivity to other antigens. Toward the development of a serological marker of exposure to Afrotropical malaria vectors we expressed the Anopheles gambiae gSG6, a small anopheline-specific salivary protein, and we measured the anti-gSG6 IgG response in individuals from a malaria hyperendemic area of Burkina Faso, West Africa. The gSG6 protein was immunogenic and anti-gSG6 IgG levels and/or prevalence increased in exposed individuals during the malaria transmission/rainy season. Moreover, this response dropped during the intervening low transmission/dry season, suggesting it is sensitive enough to detect variation in vector density. Members of the Fulani ethnic group showed higher anti-gSG6 IgG response as compared to Mossi, a result consistent with the stronger immune reactivity reported in this group. Remarkably, anti-gSG6 IgG levels among responders were high in children and gradually declined with age. This unusual pattern, opposite to the one observed with Plasmodium antigens, is compatible with a progressive desensitization to mosquito saliva and may be linked to the continued exposure to bites of anopheline mosquitoes. Overall, the humoral anti-gSG6 IgG response appears a reliable serological indicator of exposure to bites of the main African malaria vectors (An. gambiae, Anopheles arabiensis and, possibly, Anopheles funestus) and it may be exploited for malaria epidemiological studies, development of risk maps and evaluation of anti-vector measures. In addition, the gSG6 protein may represent a powerful model system to get a deeper understanding of molecular and cellular mechanisms underlying the immune tolerance and progressive desensitization to insect salivary allergens.

Background Assessment exposure and immunity to malaria is an important step in the fight against the disease. Increased malaria infection in non-immune travellers under anti-malarial chemoprophylaxis, as well as the implementation of malaria elimination programmes in endemic countries, raises new issues that pertain to these processes. Notably, monitoring malaria immunity has become more difficult in individuals showing low antibody (Ab) responses or taking medications against the Plasmodium falciparum blood stages. Commonly available techniques in malaria seroepidemiology have limited sensitivity, both against pre-erythrocytic, as against blood stages of the parasite. Thus, the aim of this study was to develop a sensitive tool to assess the exposure to malaria or to bites from the vector Anopheles gambiae, despite anti-malarial prophylactic treatment. Methods Ab responses to 13 pre-erythrocytic P. falciparum-specific peptides derived from the proteins Lsa1, Lsa3, Glurp, Salsa, Trap, Starp, CSP and Pf11.1, and to 2 peptides specific for the Anopheles gambiae saliva protein gSG6 were tested. In this study, 253 individuals from three Senegalese areas with different transmission intensities and 124 European travellers exposed to malaria during a short period of time were included. Results The multiplex assay was optimized for most but not all of the antigens. It was rapid, reproducible and required a small volume of serum. Proportions of Ab-positive individuals, Ab levels and the mean number of antigens (Ags) recognized by each individual increased significantly with increases in the level of malaria exposure. Conclusion The multiplex assay developed here provides a useful tool to evaluate immune responses to multiple Ags in large populations, even when only small amounts of serum are available, or Ab titres are low, as in case of travellers. Finally, the relationship of Ab responses with malaria endemicity levels provides a way to monitor exposure in differentially exposed autochthonous individuals from various endemicity areas, as well as in travellers who are not immune, thus indirectly assessing the parasite transmission and malaria risk in the new eradication era.

Background: Malaria and schistosomiasis coinfection frequently occurs in tropical countries. This study evaluates the influence of Schistosoma haematobium infection on specific antibody responses and cytokine production to recombinant merozoite surface protein-1-19 (MSP1-(19)) and schizont extract of Plasmodium falciparum in malaria-infected children. Methodology: Specific IgG1 to MSP1-(19), as well as IgG1 and IgG3 to schizont extract were significantly increased in coinfected children compared to P. falciparum mono-infected children. Stimulation with MSP1-(19) lead to a specific production of both interleukin-10 (IL-10) and interferon-gamma (IFN-gamma), whereas the stimulation with schizont extract produced an IL-10 response only in the coinfected group. Conclusions: Our study suggests that schistosomiasis coinfection favours anti-malarial protective antibody responses, which could be associated with the regulation of IL-10 and IFN-gamma production and seems to be antigen-dependent. This study demonstrates the importance of infectious status of the population in the evaluation of acquired immunity against malaria and highlights the consequences of a multiple infection environment during clinical trials of anti-malaria vaccine candidates.

International audience; To optimize malaria control, WHO has prioritised the need for new indicators to evaluate the efficacy of malaria vector control strategies. The gSG6-P1 peptide from gSG6 protein of Anopheles gambiae salivary glands was previously designed as a specific salivary sequence of malaria vector species. It was shown that the quantification of human antibody (Ab) responses to Anopheles salivary proteins in general and especially to the gSG6-P1 peptide was a pertinent biomarker of human exposure to Anopheles. The present objective was to validate this indicator in the evaluation of the efficacy of Insecticide Treated Nets (ITNs). A longitudinal evaluation, including parasitological, entomological and immunological assessments, was conducted on children and adults from a malaria-endemic area before and after the introduction of ITNs. Significant decrease of anti-gSG6-P1 IgG response was observed just after the efficient ITNs use. Interestingly, specific IgG Ab level was especially pertinent to evaluate a short-time period of ITNs efficacy and at individual level. However, specific IgG rose back up within four months as correct ITN use waned. IgG responses to one salivary peptide could constitute a reliable biomarker for the evaluation of ITN efficacy, at short- and long-term use, and provide a valuable tool in malaria vector control based on a real measurement of human-vector contact.

Background Intermittent preventive treatment in children (IPTc) is a promising strategy to control malaria morbidity. A significant concern is whether IPTc increases children's susceptibility to subsequent malaria infection by altering their anti-Plasmodium acquired immunity. Methods To investigate this concern, IgG antibody (Ab) responses to Plasmodium falciparum schizont extract were measured in Senegalese children (6 months-5 years old) who had received three rounds of IPTc with artesunate + sulphadoxine-pyrimethamine (or placebo) at monthly intervals eight months earlier. Potential confounding factors, such as asexual malaria parasitaemia and nutritional status were also evaluated. Results Firstly, a bivariate analysis showed that children who had received IPTc had lower anti-Plasmodium IgG Ab levels than the non-treated controls. When epidemiological parameters were incorporated into a multivariate regression, gender, nutritional status and haemoglobin concentration did not have any significant influence. In contrast, parasitaemia, past malaria morbidity and increasing age were strongly associated with a higher specific IgG response. Conclusions The intensity of the contacts with P. falciparum seems to represent the main factor influencing anti-schizont IgG responses. Previous IPTc does not seem to interfere with this parasite-dependent acquired humoral response eight months after the last drug administration.

International audience; For the fight against malaria, the World Health Organization (WHO) has emphasized the need for indicators to evaluate the efficacy of vector-control strategies. This study investigates a potential immunological marker, based on human antibody responses to Anopheles saliva, as a new indicator to evaluate the efficacy of insecticide-treated nets (ITNs). Parasitological, entomological, and immunological assessments were carried out in children and adults from a malaria-endemic region of Angola before and after the introduction of ITNs. Immunoglobulin G (IgG) levels to An. gambiae saliva were positively associated with the intensity of An. gambiae exposure and malaria infection. A significant decrease in the anti-saliva IgG response was observed after the introduction of ITNs, and this was associated with a drop in parasite load. This study represents the first stage in the development of a new indicator to evaluate the efficacy of malaria vector-control strategies, which could apply in other arthropod vector-borne diseases.

Background In sub-Saharan Africa, preschool children represent the population most vulnerable to malaria and malnutrition. It is widely recognized that malnutrition compromises the immune function, resulting in higher risk of infection. However, very few studies have investigated the relationship between malaria, malnutrition and specific immunity. In the present study, the anti-Plasmodium falciparum IgG antibody (Ab) response was evaluated in children according to the type of malnutrition. Methods Anthropometric assessment and blood sample collection were carried out during a cross-sectional survey including rural Senegalese preschool children. This cross-sectional survey was conducted in July 2003 at the onset of the rainy season. Malnutrition was defined as stunting (height-for-age P. falciparum whole extracts (schizont antigens) was assessed by ELISA in sera of the included children. Results Both the prevalence of anti-malarial immune responders and specific IgG Ab levels were significantly lower in malnourished children than in controls. Depending on the type of malnutrition, wasted children and stunted children presented a lower specific IgG Ab response than their respective controls, but this difference was significant only in stunted children (P = 0.026). This down-regulation of the specific Ab response seemed to be explained by severely stunted children (HAZ ≤ -2.5) compared to their controls (P = 0.03), while no significant difference was observed in mildly stunted children (-2.5 < HAZ P. falciparum Ab response appeared to be independent of the intensity of infection. Conclusion Child malnutrition, and particularly stunting, may down-regulate the anti-P. falciparum Ab response, both in terms of prevalence of immune responders and specific IgG Ab levels. This study provides further evidence for the influence of malnutrition on the specific anti-malarial immune response and points to the importance of taking into account child malnutrition in malaria epidemiological studies and vaccine trials.

Background Human populations exposed to low malaria transmission present particular severe risks of malaria morbidity and mortality. In addition, in a context of low-level exposure to Anopheles vector, conventional entomological methods used for sampling Anopheles populations are insufficiently sensitive and probably under-estimate the real risk of malaria transmission. The evaluation of antibody (Ab) responses to arthropod salivary proteins constitutes a novel tool for estimating exposure level to insect bites. In the case of malaria, a recent study has shown that human IgG responses to the gSG6-P1 peptide represented a specific biomarker of exposure to Anopheles gambiae bites. The objective of this study was to investigate if this biomarker can be used to estimate low-level exposure of individuals to Anopheles vector. Methods The IgG Ab level to gSG6-P1 was evaluated at the peak and at the end of the An. gambiae exposure season in children living in Senegalese villages, where the Anopheles density was estimated to be very low by classical entomological trapping but where malaria transmission occurred during the studied season. Results Specific IgG responses to gSG6-P1 were observed in children exposed to very low-level of Anopheles bites. In addition, a significant increase in the specific IgG Ab level was observed during the Anopheles exposure season whereas classical entomological data have reported very few or no Anopheles during the studied period. Furthermore, this biomarker may also be applicable to evaluate the heterogeneity of individual exposure. Conclusion The results strengthen the hypothesis that the evaluation of IgG responses to gSG6-P1 during the season of exposure could reflect the real human contact with anthropophilic Anopheles and suggest that this biomarker of low exposure could be used at the individual level. This promising immuno-epidemiological marker could represent a useful tool to assess the risk to very low exposure to malaria vectors as observed in seasonal, urban, altitude or travellers contexts. In addition, this biomarker could be used for the surveillance survey after applying anti-vector strategy.

International audience; Background: n order to improve malaria control, and under the aegis of WHO recommendations, many efforts are being devoted to developing new tools for identifying geographic areas with high risk of parasite transmission. Evaluation of the human antibody response to arthropod salivary proteins could be an epidemiological indicator of exposure to vector bites, and therefore to risk of pathogen transmission. In the case of malaria, which is transmitted only by anopheline mosquitoes, maximal specificity could be achieved through identification of immunogenic proteins specific to the Anopheles genus. The objective of the present study was to determine whether the IgG response to the Anopheles gambiae gSG6 protein, from its recombinant form to derived synthetic peptides, could be an immunological marker of exposure specific to Anopheles gambiae bites.Methodology/Principal Findings: Specific IgG antibodies to recombinant gSG6 protein were observed in children living in a Senegalese area exposed to malaria. With the objective of optimizing Anopheles specificity and reproducibility, we designed five gSG6-based peptide sequences using a bioinformatic approach, taking into consideration i) their potential antigenic properties and ii) the absence of cross-reactivity with protein sequences of other arthropods/organisms. The specific anti-peptide IgG antibody response was evaluated in exposed children. The five gSG6 peptides showed differing antigenic properties, with gSG6-P1 and gSG6-P2 exhibiting the highest antigenicity. However, a significant increase in the specific IgG response during the rainy season and a positive association between the IgG level and the level of exposure to Anopheles gambiae bites was significant only for gSG6-P1.Conclusions/Significance: This step-by-step approach suggests that gSG6-P1 could be an optimal candidate marker for evaluating exposure to Anopheles gambiae bites. This marker could be employed as a geographic indicator, like remote sensing techniques, for mapping the risk of malaria. It could also represent a direct criterion of efficacy in evaluation of vector control strategies

Background In sub-Saharan areas, malaria transmission was mainly ensured by Anopheles. gambiae s.l. and Anopheles. funestus vectors. The immune response status to Plasmodium falciparum was evaluated in children living in two villages where malaria transmission was ensured by dissimilar species of Anopheles vectors (An. funestus vs An. gambiae s.l.). Methods A multi-disciplinary study was performed in villages located in Northern Senegal. Two villages were selected: Mboula village where transmission is strictly ensured by An. gambiae s.l. and Gankette Balla village which is exposed to several Anopheles species but where An. funestus is the only infected vector found. In each village, a cohort of 150 children aged from one to nine years was followed during one year and IgG response directed to schizont extract was determined by ELISA. Results Similar results of specific IgG responses according to age and P. falciparum infection were observed in both villages. Specific IgG response increased progressively from one-year to 5-year old children and then stayed high in children from five to nine years old. The children with P. falciparum infection had higher specific antibody responses compared to negative infection children, suggesting a strong relationship between production of specific antibodies and malaria transmission, rather than protective immunity. In contrast, higher variation of antibody levels according to malaria transmission periods were found in Mboula compared to Gankette Balla. In Mboula, the peak of malaria transmission was followed by a considerable increase in antibody levels, whereas low and constant anti-malaria IgG response was observed throughout the year in Gankette Balla. Conclusion This study shows that the development of anti-malaria antibody response was profoundly different according to areas where malaria exposure is dependent with different Anopheles species. These results are discussed according to i) the use of immunological tool for the evaluation of malaria transmission and ii) the influence of Anopheles vectors species on the regulation of antibody responses to P. falciparum.

Background During blood feeding, the mosquito injects saliva into the vertebrate host. This saliva contains bioactive components which may play a role in pathogen transmission and in host-vector relationships by inducing an immune response in the vertebrate host. The evaluation of human immune responses to arthropod bites might also represent a research direction for assessing individual exposure to the bite of a malaria vector. Methods The present study examined the antibody (Ab) IgG response during the season of exposure to Anopheles gambiae bites in young children living in a malaria endemic area. Immunoblots were performed with An. gambiae saliva to detect anti-saliva Ab bands and the evolution of immunogenic bands at the peak of, and following, the transmission period. Results The results showed that anti-Anopheles Ab was directed against a limited number of salivary proteins (175, 115, 72 and 30 kDa bands). Specific IgG responses to mosquito salivary proteins were variable among exposed individuals; nevertheless, two major bands (175 and 72 kDa) were observed in all immune-responder children. Analysis of the intensity of immunogenic bands revealed that IgG levels against the 175 kDa band were significantly higher during the peak period compared to the end period malaria transmission. Conclusion This preliminary work supports the potential of using anti-saliva immune responses as a measure of exposure to Anopheles bites. The use of immunoblots coupled with evaluation of band intensity could be an adequate tool for distinguishing immunogenic salivary proteins as candidate markers of bite exposure. Furthermore, this study may open the way to design new epidemiological tools for evaluating the risk of malaria exposure.

Following the implementation of two dams in the Senegal River, entomological and parasitological studies were conducted in three different ecological zones in the Senegal River Basin (the low valley of Senegal River, the Guiers Lake area and the low valley of Ferlo) every 3 month in June 2004, September 2004, December 2004 and March 2005. The objective of this work was to study the influence of environmental heterogeneities on vector bionomics and malaria epidemiology. Mosquitoes were collected when landing on human volunteers and by pyrethrum spray catches. In the parasitological survey, blood samples were taken from a cohort of schoolchildren under 9 years during each entomology survey. Seven anopheline species were collected: Anopheles arabiensis, Anopheles gambiae M form, Anopheles funestus, Anopheles pharoensis, Anopheles coustani, Anopheles wellcomei and Anopheles rufipes. A. arabiensis, A. funestus and A. pharoensis were predominant in the low valley of the Senegal River, A. funestus in the Guiers Lake area and A. arabiensis in the low valley of Ferlo. Mosquito populations' dynamics varied temporally depending on the rainy season for each zone. The anthropophilic rates varied between 6 and 76% for A. gambiae s.l. and 23 and 80% for A. funestus. Only 4/396 A. pharoensis and 1/3076 A. funestus tested carried Plasmodium falciparum CS antigen. These results suggest the implication of A. pharoensis in malaria transmission. The related entomological inoculation rates were estimated to 10.44 in Mbilor and 3 infected bites in Gankette Balla and were due, respectively, to A. pharoensis and A. funestus. Overall, 1636 thick blood smears were tested from blood samples taken from schoolchildren with, respectively, a parasite and gametocyte average prevalence of 9 and 0.9%. The parasite prevalence was uniformly low in Mbilor and Gankette Balla whereas; it increased in September (16%) and then remained stable in December and March (22%) in Mboula where malaria transmission was not perceptible. However, significant differences were observed over time for parasite prevalence in Mbilor and Mboula villages whereas; it was only in Gankette Balla village where gametocyte prevalence was significantly different over time. Our study demonstrates the influence of ecological changes resulted from dams implementation in the Senegal River on the composition of vectorial system, malaria transmission and epidemiology. Such changes should be thoroughly surveyed in order to prevent any possible malaria outbreak in the Senegal River Basin.

International audience; Artesunate is a highly effective antimalarial. and there is some evidence that it is also active against schistosome infections. We therefore investigated whether treatment with artesunate of acute malaria in Senegalese children had an impact on their level of infection with Schistosoma haematobium. Twenty-seven children who were entered into a clinical trial of antimalaria treatment were excreting S. haematobium eggs in their urine on the day of treatment. Fifteen children received a combination of a single dose of sulfadoxine/pyrimethamine together with three daily doses of artesunate (4 mg/kg); the remaining 12 children received three daily doses of amodiaquine and artesunate. The overall cure rate and reduction in the mean number of excreted eggs at 28 days post treatment were 92.6% and 94.5%, respectively. Our findings indicate that artesunate, in addition to being a very effective treatment for uncomplicated malaria, can also sharply reduce the S. haematobium loads harboured by pre-school African children. (C) 2006 Published by Elsevier Ltd on behalf of Royal Society of Tropical Medicine and Hygiene.

Exposure to vectors of infectious diseases has been associated with antibody responses against salivary antigens of arthropods among people living in endemic areas. This immune response has been proposed as a surrogate marker of exposure to vectors appropriate for evaluating the protective efficacy of antivectorial devices. The existence and potential use of such antibody responses in travellers transiently exposed to Plasmodium or arbovirus vectors in tropical areas has never been investigated. The IgM and IgG antibody responses of 88 French soldiers against the saliva of Anopheles gambiae and Aedes aegypti were evaluated before and after a 5-month journey in tropical Africa. Antibody responses against Anopheles and Aedes saliva increased significantly in 41% and 15% of the individuals, respectively, and appeared to be specific to the mosquito genus. A proteomic and immunoproteomic analysis of anopheles and Aedes saliva allowed for the identification of some antigens that were recognized by most of the exposed individuals. These results suggest that antibody responses to the saliva of mosquitoes could be considered as specific surrogate markers of exposure of travellers to mosquito vectors that transmit arthropod borne infections.

International audience; The morbidity and mortality of vector-borne diseases is closely linked to exposure of the human host to vectors. Qualitative and quantitative evaluation of individual exposure to arthropod bites by investigation of the specific immune response to vector saliva would make it possible to monitor individuals at risk of vectorial transmission of pathogens. The objective of this study was to evaluate and compare the antibody (IgG) response to saliva from uninfected Glossina species, vectors, or non-vectors of Trypanosoma brucei gambiense by detecting immunogenic proteins in humans residing in an area endemic for human African trypanosomiasis in the Democratic Republic of Congo. Our results suggest that the immunogenic profiles observed seemed specific to the Glossina species (vector or non-vector species) and to the infectious status of exposed individuals (infected or not infected). This preliminary work tends to support the feasibility of development of an epidemiologic tool based on this antibody response to salivary proteins.