Emerging knock-down resistance in Anopheles arabiensis populations of Dakar, Senegal: first evidence of a high prevalence of kdr-e mutation in West African urban area.

BACKGROUND: Urban malaria is now considered a major emerging health problem in Africa and urban insecticide resistance may represent a serious threat to the ambitious programme of further scaling-up coverage with long-lasting insecticide-treated bed nets and indoor residual spray. This study evaluates the levels and mechanisms of insecticide resistance in Anopheles gambiae populations in 44 urban areas of Dakar in a longitudinal entomological surveillance study. METHODS: Adult mosquitoes sampled by night-landing catches at 44 sites across Dakar from 2007 to 2010 were genotyped to assess the frequency and distribution of resistance alleles. In addition World Health Organization susceptibility tests to six insecticides were performed on F0 adults issuing from immature stages of An. gambiae s.l. sampled in August 2010, 2011 and 2012 in three sites of Dakar: Pikine, Thiaroye and Almadies and repeated in 2012 with three of the insecticides after PBO exposure to test for mechanisms of oxydase resistance. Species, molecular forms and the presence of kdr and ace-1 mutations were assessed by polymerase chain reaction. RESULTS: High frequencies of the kdr-e allele, ranging from 35 to 100 %, were found in Anopheles arabiensis at all 44 sites. The insecticide susceptibility tests indicated sensitivity to bendiocarb in Almadies in 2010 and 2011 and in Yarakh between 2010 and 2012 and sensitivity to fenitrothion in Almadies in 2010. The mortality rate of EE genotype mosquitoes was lower and that of SS mosquitoes was higher than that of SE mosquitoes, while the mortality rate of the SW genotype was slightly higher than that of the SE genotype. Pyperonyl butoxide (PBO) had a significant effect on mortality in Pikine (OR = 1.4, 95 % CI = 1.3-1.5, with mortality of 42-55 % after exposure and 11-17 % without PBO) and Yarakh (OR = 1.6, 95 % CI = 1.4-1.7, with mortality of 68-81 % after exposure and 23-37 % without), but not in Almadies (OR = 1.0, 95 % CI = 0.9-1.1). CONCLUSION: A high prevalence of kdr-e in West Africa was demonstrated, and knock-down resistance mechanisms predominate although some oxidases mechanisms (cytochrome P450 monooxygenases) also occur. In view of the increased use of insecticides and the proposed role of the kdr gene in the susceptibility of Anopheles to Plasmodium, this finding will significantly affect the success of vector control programmes.

Relationship between exposure to vector bites and antibody responses to mosquito salivary gland extracts.

Mosquito-borne diseases are major health problems worldwide. Serological responses to mosquito saliva proteins may be useful in estimating individual exposure to bites from mosquitoes transmitting these diseases. However, the relationships between the levels of these IgG responses and mosquito density as well as IgG response specificity at the genus and/or species level need to be clarified prior to develop new immunological markers to assess human/vector contact. To this end, a kinetic study of antibody levels against several mosquito salivary gland extracts from southeastern French individuals living in three areas with distinct ecological environments and, by implication, distinct Aedes caspius mosquito densities were compared using ELISA. A positive association was observed between the average levels of IgG responses against Ae. caspius salivary gland extracts and spatial Ae. caspius densities. Additionally, the average level of IgG responses increased significantly during the peak exposure to Ae. caspius at each site and returned to baseline four months later, suggesting short-lived IgG responses. The species-specificity of IgG antibody responses was determined by testing antibody responses to salivary gland extracts from Cx. pipiens, a mosquito that is present at these three sites at different density levels, and from two other Aedes species not present in the study area (Ae. aegypti and Ae. albopictus). The IgG responses observed against these mosquito salivary gland extracts contrasted with those observed against Ae. caspius salivary gland extracts, supporting the existence of species-specific serological responses. By considering different populations and densities of mosquitoes linked to environmental factors, this study shows, for the first time, that specific IgG antibody responses against Ae. caspius salivary gland extracts may be related to the seasonal and geographical variations in Ae. caspius density. Characterisation of such immunological-markers may allow the evaluation of the effectiveness of vector-control strategies or estimation of the risk of vector-borne disease transmission.

Assessment of exposure to Plasmodium falciparum transmission in a low endemicity area by using multiplex fluorescent microsphere-based serological assays.

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.

Determinants of compliance with anti-vectorial protective measures among non-immune travellers during missions to tropical Africa.

BACKGROUND: The effectiveness of anti-vectorial malaria protective measures in travellers and expatriates is hampered by incorrect compliance. The objective of the present study was to identify the determinants of compliance with anti-vectorial protective measures (AVPMs) in this population that is particularly at risk because of their lack of immunity. METHODS: Compliance with wearing long clothing, sleeping under insecticide-impregnated bed nets (IIBNs) and using insect repellent was estimated and analysed by questionnaires administered to 2,205 French military travellers from 20 groups before and after short-term missions (approximately four months) in six tropical African countries (Senegal, Ivory Coast, Chad, Central African Republic, Gabon and Djibouti). For each AVPM, the association of "correct compliance" with individual and collective variables was investigated using random-effect mixed logistic regression models to take into account the clustered design of the study. RESULTS: The correct compliance rates were 48.6%, 50.6% and 18.5% for wearing long clothing, sleeping under bed nets and using repellents, respectively. Depending on the AVPM, correct compliance was significantly associated with the following factors: country, older than 24 years of age, management responsibilities, the perception of a personal malaria risk greater than that of other travellers, the occurrence of life events, early bedtime (i.e., before midnight), the type of stay (field operation compared to training), the absence of medical history of malaria, the absence of previous travel in malaria-endemic areas and the absence of tobacco consumption.There was no competition between compliance with the different AVPMs or between compliance with any AVPM and malaria chemoprophylaxis. CONCLUSION: Interventions aimed at improving compliance with AVPMs should target young people without management responsibilities who are scheduled for non-operational activities in countries with high risk of clinical malaria. Weak associations between compliance and history of clinical malaria or variables that pertain to threat perception suggest that cognition-based interventions referencing a "bad experience" with clinical malaria could have only a slight impact on the improvement of compliance. Further studies should focus on the cognitive and behavioural predictors of compliance with AVPMs.

A multiplex assay for the simultaneous detection of antibodies against 15 Plasmodium falciparum and Anopheles gambiae saliva antigens.

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.

Determinants of compliance with malaria chemoprophylaxis among French soldiers during missions in inter-tropical Africa.

BACKGROUND: The effectiveness of malaria chemoprophylaxis is limited by the lack of compliance whose determinants are not well known. METHODS: The compliance with malaria chemoprophylaxis has been estimated and analysed by validated questionnaires administered before and after the short-term missions (about four months) in five tropical African countries of 2,093 French soldiers from 19 military companies involved in a prospective cohort study. "Correct compliance" was defined as "no missed doses" of daily drug intake during the entire mission and was analysed using multiple mixed-effect logistic regression model. RESULTS: The averaged prevalence rate of correct compliance was 46.2%, ranging from 9.6%to 76.6% according to the companies. Incorrect compliance was significantly associated with eveningness (p = 0.028), a medical history of clinical malaria (p < 0.001) and a perceived mosquito attractiveness inferior or superior to the others (p < 0.007). Correct compliance was significantly associated with the systematic use of protective measures against mosquito bites (p < 0.001), the type of military operations (combat vs. training activities, p < 0.001) and other individual factors (p < 0.05). CONCLUSIONS: The identification of circumstances and profiles of persons at higher risk of lack of compliance would pave the way to specifically targeted strategies aimed to improve compliance with malaria chemoprophylaxis and, therefore, its effectiveness.

Salivary gland protein repertoire from Aedes aegypti mosquitoes.

Diseases caused by arthropod-borne viruses are a significant threat to the health of human and animal populations throughout the world. Better knowledge of the molecules synthesized in the salivary gland and saliva of hematophagous arthropods could be of use for improving the control of pathogen transmission. Recently, a sialome analysis of three Aedes aegypti mosquito colonies (PAEA, Rockefeller, and Formosus) carried out in our laboratory allowed us to identify 44 saliva proteins. Of these secreted proteins, none was exclusively expressed in one colony, suggesting that expression of salivary proteins is highly conserved across populations. In another study, we reported that some of these salivary proteins could be used as the genus-specific markers for travelers' exposure to mosquito vectors. Here, comparison of salivary gland protein profiles between these same three Ae. aegypti colonies was performed using the one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) difference gel electrophoresis method. As observed at the saliva level, no significant differences were detected between these three colonies. The salivary gland protein repertoire from the Ae. aegypti mosquito was analyzed using a proteomic approach. One hundred and twenty proteins were identified in these salivary glands representing the largest description of the Ae. aegypti salivary gland protein catalog. We succeeded in identifying 15 secreted proteins, some of which have already been reported as being involved in blood feeding. A comparison of the proteins identified between the salivary glands and the sialome is discussed.

Major variations in malaria exposure of travellers in rural areas: an entomological cohort study in western Côte d'Ivoire.

BACKGROUND: Malaria remains a major threat, to both travellers and military personnel deployed to endemic areas. The recommendations for travellers given by the World Health Organization is based on the incidence of malaria in an area and do not take the degree of exposure into account. The aim of this article is to evaluate the exposure of travellers by entomologic methods, which are the commonly used measures of the intensity of malaria transmission. METHODS: From February 2004 to June 2004, five groups of 30 military personnel were stationed in up to 10 sites in western Côte d'Ivoire, from one week to several months. Adult mosquitoes were collected by human landing catches at each site during the five months and the level of exposure to malaria transmission of each group was estimated. RESULTS: The level of transmission varied from one site to another one from less than one to approximately more than 100 infective bites per month. In the majority of sites, at least two anopheline species were involved in transmission. The cumulative EIR over the study period varied according to the groups from 29 infected bites per person/per mission to 324. CONCLUSION: The level of malaria transmission and malaria risk varies widely (varying by a factor of eleven) between groups of travellers travelling in the same region and at the same time. Physicians involved in travel medicine or supporting expatriated populations or refugees should consider this heterogeneity and emphasize the importance of combining appropriate measures, such as chemoprophylaxis and protective measures against mosquitoes.

Remote sensing and malaria risk for military personnel in Africa.

BACKGROUND: Nonimmune travelers in malaria-endemic areas are exposed to transmission and may experience clinical malaria attacks during or after their travel despite using antivectorial devices or chemoprophylaxis. Environment plays an essential role in the epidemiology of this disease. Remote-sensed environmental information had not yet been tested as an indicator of malaria risk among nonimmune travelers. METHODS: A total of 1,189 personnel from 10 French military companies traveling for a short-duration mission (about 4 mo) in sub-Saharan Africa from February 2004 to February 2006 were enrolled in a prospective longitudinal cohort study. Incidence rate of clinical malaria attacks occurring during or after the mission was analyzed according to individual characteristics, compliance with antimalaria prophylactic measures, and environmental information obtained from earth observation satellites for all the locations visited during the missions. RESULTS: Age, the lack of compliance with the chemoprophylaxis, and staying in areas with an average Normalized Difference Vegetation Index higher than 0.35 were risk factors for clinical malaria. CONCLUSIONS: Remotely sensed environmental data can provide important planning information on the likely level of malaria risk among nonimmune travelers who could be briefly exposed to malaria transmission and could be used to standardize for the risk of malaria transmission when evaluating the efficacy of antimalaria prophylactic measures.

Antibody response against saliva antigens of Anopheles gambiae and Aedes aegypti in travellers in tropical Africa.

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.

[Antiparasitic vaccines: where are we now?]. / Vaccins antiparasitaires: où en est-on ?

In more than one way, the development of antiparasitic vaccines is challenging, but major efforts have been made. A large number of clinical trials have been carried out and a few antigens have been tested in the endemic zone, especially against malaria. So far, no vaccine candidate has shown a sufficient and long-lasting effectiveness that would be useful for public health. However, the trials have shown without ambiguity that a certain level of clinical immunity against paludism, schistosomiasis or leishmaniasis could be induced by vaccination, in the experimental setting or in the field.

Malaria transmission and insecticide resistance of Anopheles gambiae (Diptera: Culicidae) in the French military camp of Port-Bouët, Abidjan (Côte d'Ivoire): implications for vector control.

An important vector control program is ongoing to lower the risk of malaria transmission in the French military camp of Port-Bouët, Abidjan (Côte d'Ivoire). However, some autochthonous malaria cases are regularly suspected. An entomological survey was conducted in June 2004 in the camp to assess malaria transmission and evaluate the pyrethroid and organophosphate resistance of the malaria vectors. The average mosquito biting rate was 178.0 bites per person per night. Mosquitoes belonging to the Anopheles gambiae (Diptera: Culicidae) complex and the Anopheles funestus group were collected. An. gambiae s.s. molecular form M was the only species of the An. gambiae complex present. The average number of An. gambiae bites was approximately 44.3 per person per night. The circumsporozoite index was 0.38% and the entomological inoculation rate estimated to be 1.2 infective bites per week for the study period. The kdr and ace1 gene frequencies in the An. gambiae population were 0.70 and 0.15, respectively. Personnel living in the French barracks of Port-Bouët are thus at high risk of being bitten by parasite-infected mosquitoes. Such an entomological inoculation rate, usually found in African peri-urban environments, was unexpected considering the extensive effort deployed to control mosquitoes in the camp. Insecticide resistance could explain the inefficacy of the vector control program but the spraying strategy is also questionable.

Chloroquine resistance reversal agents as promising antimalarial drugs.

The development and spread of resistance to antimalarial drugs poses a severe and increasing public health threat. Failures of prophylaxis or treatment with quinolines, hydroxynaphthoquinones, sesquiterpene lactones, antifolate drugs and sulfamides are involved in a return malaria-related morbidity and mortality. Resistance is associated with a decrease in accumulation of drugs into the vacuole, which results from a reduced uptake of the drug, an increased efflux or a combination of both. A number of candidate genes in P. falciparum have been proposed to be involved in antimalarial resistance, each concerned in membrane transport. Weaker or stronger associations are seen in P. falciparum between the resistance to quinolines or artemisinin derivatives and codon changes in Pfmdr1, a gene which encodes Pgh-1, an ortholog of one of the P-glycoproteins expressed in multi-drug resistant human cancer cells (ABC transporter). Further analysis has revealed a new gene, Pfcrt, encoding a PfCRT protein, which resembles an anion channel. Codon changes found in the Pfcrt sequence in drug resistant isolates could facilitate the drug efflux through a putative channel. It has been proposed that the reversal of quinoline resistance by verapamil is due to hydrophobic binding to the mutated PfCRT protein. Several compounds have demonstrated in the past decade a promising capability to reverse the antimalarial drug resistance in vitro in parasite isolates, in animal models and in human malaria. These drugs belong to different pharmacological classes such as calcium channel blockers, tricyclic antidepressants, antipsychotic calmodulin antagonists, histamine H1-receptor antagonists, analgesic and antipyretic drugs, non-steroidal anti-inflammatory drugs, and to different chemical classes such as synthetic surfactants, alkaloids from plants used in traditional medicine, pyrrolidinoaminoalkanes and anthracenic derivatives. Here we summarize the progress made in biochemical and genetic basis of antimalarial resistance, emphasizing the recent developments on drugs, which interfere with trans membrane proteins involved in drug efflux or uptake.

Antibody responses to several malaria pre-erythrocytic antigens as a marker of malaria exposure among travelers.

Protective devices against vectors are used by travelers in malaria-endemic areas but their efficacy for protection against mosquitoes has rarely been evaluated. The level of exposure to malaria transmission of 205 soldiers deployed in Africa and the efficacy of their anti-vector prophylaxis was evaluated by comparison of their IgM and IgG responses against five pre-erythrocytic Plasmodium falciparum antigens (circumsporozoite protein, sporozoite threonine- and asparagine-rich protein, sporozoite- and liver-stage antigen, liver stage antigen 1, and SR11.1) before and at the end of their deployment, and three months after returning to France for 106 of these soldiers. The immune responses increased significantly during the mission in 35% (95% confidence interval = 28-42%) of the individuals. The permanent use of insecticide-treated bed nets and long-sleeve battle dress at night were associated with protective efficacy. The analysis of these antibody responses was sensitive enough to evaluate exposure to malaria transmission and the efficacy of anti-vector devices in travelers using antimalarial chemoprophylaxis.