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Although the urban areas of Abidjan, Cote d'Ivoire have faced recurrent outbreaks of Aedes-borne arboviruses, the seasonal dynamics of local populations of the key vector Aedes aegypti remained sti...

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

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 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.

Offianan Andre Toure,1 Tiacoh N’Guessan Landry,1 Serge Brice Assi,2,3 Antoinette Amany Kone,1Eric Adji Gbessi,1Berenger Aristide Ako,1 Baba Coulibaly,1 Bouakary Kone,4 Oumar Ouattara,4 Sylvain Beourou,1 Alphonsine Koffi,2 Franck Remoue,2,5 Christophe Rogier6 1Malariology Unit, Pasteur Institute of Côte d’Ivoire, Abidjan, Côte d’Ivoire; 2Malaria and Anopheles Research and Management Unit, Pierre Richet Institute, Bouake, Côte d’Ivoire; 3National Malaria Control Program, Bouake, Côte d’Ivoire; 4Department of Medicine, Health Care Center of Dar-Es-Salam, Bouake, Côte d’Ivoire; 5UMR 224-MIVEGEC, Research Development Institute, Montpellier, France; 6Army Health Department, Paris, France Introduction: Parasite clearance is useful to detect artemisinin resistance. The aim of this study was to investigate parasite clearance in patients treated with artesunate + amodiaquine (AS + AQ) and artemether + lumefantrine (AL): the two artemisinin-based combination therapies (ACTs) recommended in the first-line treatment of uncomplicated malaria in Côte d’Ivoire.Methods: This study was conducted in Bouaké, Côte d’Ivoire, from April to June 2016. Patients aged at least 6 months with uncomplicated malaria and treated with AS + AQ or AL were hospitalized for 3 days, and follow-up assessments were performed on days 3, 7, 14, 21, 28, 35, and 42. Blood smears were collected at the time of screening, pre-dose, and 6-hour intervals following the first dose of administration until two consecutive negative smears were recorded, thereafter at day 3 and follow-up visits. Parasite clearance was determined using the Worldwide Antimalarial Resistance Network’s parasite clearance estimator. The primary end points were parasite clearance rate and time.Results: A total of 120 patients (57 in the AS + AQ group and 63 in the AL group) were randomized among 298 patients screened. The median parasite clearance time was 30 hours (IQR, 24–36 hours), for each ACT. The median parasite clearance rate had a slope half-life of 2.36 hours (IQR, 1.85–2.88 hours) and 2.23 hours (IQR, 1.74–2.63 hours) for AS + AQ and AL, respectively. The polymerase chain reaction-corrected adequate clinical and parasitological response was 100% and 98.07% at day 42 for AS + AQ and AL, respectively.Conclusion: Patients treated with AS + AQ and AL had cleared parasites rapidly. ACTs are still efficacious in Bouaké, Côte d’Ivoire, but continued efficacy monitoring of ACTs is needed. Keywords: Plasmodium falciparum, ACTs, parasite clearance, Côte d’Ivoire

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.

Objective Using human IgG antibody response to the Aedes Nterm-34 kDa salivary peptide as an indicator of human exposure to Aedes bites in surveying exposed populations from areas at risk of dengue virus (DENV) transmission in urban settings of Vientiane city, Lao PDR. Methods Enzyme-linked immunosorbent assay tests were performed to measure the IgG response to Nterm-34 kDa peptide in blood samples collected within a flavivirus seroprevalence survey carried out in 2006 including 3558 randomly selected individuals. The level of IgG response to the Nterm-34 kDa peptide in individuals was analysed in relation to the level of urbanisation of the individual's residence, areas that presented significant differences in the prevalence of recent DENV infection. Results No differences were observed in the anti-Nterm-34 kDa IgG level between DENV-positive and DENV-negative individuals. However, the level of specific IgG response was higher among individuals living in slightly urbanised neighbourhoods than among those in more highly urbanised areas (P

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

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.

Arthropod-borne viruses (arboviruses) such as dengue virus (DENV), chikungunya virus (CHIKV), Zika virus (ZIKV), and yellow fever virus (YFV) are the most important ‘emerging pathogens’ because of their geographic spread and their increasing impact on vulnerable human populations. To fight against these arboviruses, vector control strategies (VCS) remain one of the most valuable means. However, their implementation and monitoring are labour intensive and difficult to sustain on large scales, especially when transmission and Aedes mosquito densities are low. To increase the efficacy of VCS, current entomological methods should be improved by new complementary tools which measure the risk of arthropod-borne diseases’ transmission. The study of human–Aedes immunological relationships can provide new promising serological tools, namely antibody-based biomarkers, allowing to accurately estimate the human–Aedes contact and consequently, the risk of transmission of arboviruses and the effectiveness of VCS. This review focuses on studies highlighting the concept, techniques, and methods used to develop and validate specific candidate biomarkers of human exposure to Aedes bites. Potential applications of such antibody-based biomarkers of exposure to Aedes vector bites in the field of operational research are also discussed.

Summary Objective The development of a biomarker of exposure based on the evaluation of the human antibody response specific to Anopheles salivary proteins seems promising in improving malaria control. The IgG response specific to the gSG6-P1 peptide has already been validated as a biomarker of An. gambiae exposure. This study represents a first attempt to validate the gSG6-P1 peptide as an epidemiological tool evaluating exposure to An. funestus bites, the second main malaria vector in sub-Saharan Africa. Methods A multi-disciplinary survey was performed in a Senegalese village where An. funestus represents the principal anopheline species. The IgG antibody level specific to gSG6-P1 was evaluated and compared in the same children before, at the peak and after the rainy season. Results Two-thirds of the children developed a specific IgG response to gSG6-P1 during the study period and – more interestingly – before the rainy season, when An. funestus was the only anopheline species reported. The specific IgG response increased during the An. funestus exposure season, and a positive association between the IgG level and the level of exposure to An. funestus bites was observed. Conclusions The results suggest that the evaluation of the IgG response specific to gSG6-P1 in children could also represent a biomarker of exposure to An. funestus bites. The availability of such a biomarker evaluating the exposure to both main Plasmodium falciparum vectors in Africa could be particularly relevant as a direct criterion for the evaluation of the efficacy of vector control strategies. Premiere tentative de validation du peptide salivaire gSG6-P1 comme un outil immuno-epidemiologique d’evaluation de l’exposition humaine aux piqures de An. funestus Objectif: Valider le peptide gSG6-P1 comme un outil epidemiologique evaluant l’exposition aux piqures de An. funestus, le second principal vecteur de la malaria en Afrique subsaharienne. Methodes: Etude multidisciplinaire dans un village senegalais ouAn. funestus est la principale espece d’anophele. Le taux d’anticorps IgG specifiques a gSG6-P1 a eteevalue et compare chez les memes enfants avant, durant le pic et a la fin de la saison des pluies. Resultats: Deux tiers des enfants ont developpe une reponse IgG specifique a gSG6-P1 durant la periode etudiee et - plus interessant - avant la saison des pluies, quand An. funestus etait la seule espece d’anophele signalee. La reponse IgG specifique augmentait au cours de saison d’exposition a l’An. funestus et une association positive entre le taux d’IgG et le niveau d’exposition aux piqures de An. funestus a ete observee. Conclusions: Les resultats suggerent que l’evaluation de la reponse IgG specifique a gSG6-P1 chez les enfants pourrait egalement representer un biomarqueur de l’exposition aux piqures de An. funestus. La disponibilite d’un tel biomarqueurs evaluant l’exposition aux deux principaux vecteurs de P. falciparum en Afrique pourrait etre particulierement pertinente en tant que critere pour l’evaluation directe de l’efficacite des strategies de lutte antivectorielle. Primer ensayo para validar el peptido salivar gSG6-P1 como herramienta inmuno-epidemiologica que evalue la exposicion humana frente a picaduras de An. funestus Objetivo: Validar el peptido gSG6-P1 como herramienta epidemiologica que evalue la exposicion a picaduras de An. funestus, el segundo vector de malaria mas importante en Africa sub-Sahariana. Metodos: Estudio multidisciplinar, en un poblado senegales en donde An. funestus es la principal especie anofelina. Los niveles de anticuerpos IgG especificos para gSG6-P1 se evaluaron y compararon en los mismos ninos antes, durante el pico y despues de la estacion de lluvias. Resultados: Dos tercios de los ninos desarrollaron una IgG especifica para gSG6-P1 durante el periodo de estudio y – mas interesante aun – antes de la epoca lluviosa, cuando An. funestus era la unica especie anofelina reportada. La respuesta IgG especifica aumento durante el periodo de exposicion a An. funestus y se observo una asociacion positiva entre el nivel de IgG y el nivel de exposicion a picaduras de An. funestus. Conclusiones: Los resultados sugieren que la evaluacion de la respuesta IgG especifica para gSG6-P1 en ninos tambien podria utilizarse como un biomarcador de exposicion a picaduras de An. funestus. La disponibilidad de un biomarcador que evalua la exposicion a los dos principales vectores de P. falciparum en Africa podria ser particularmente relevante como un criterio directo para la evaluacion de la eficacia de estrategias de control vectorial.

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.

Evaluation of vector control is crucial for improving malaria containment and, according to World Health Organization, new complementary indicators would be very valuable. In this study the IgG response to the Anopheles-specific cE5 salivary protein was tested as a tool to evaluate the efficacy of insecticide-treated nets in reducing human exposure to malaria vectors. Sera collected during a longitudinal study carried out in Angola, and including entomological and parasitological data, were used to assess the IgG response to the Anopheles gambiae cE5 in both children and adults, before and after the application of insecticide-treated nets. Seasonal fluctuation of specific IgG antibody levels according to exposure was only found in children (up to approximate to 14 years old) whose anti-cE5 IgG response dropped after bed nets installation. These results were fully consistent with previous findings obtained with the same set of sera and indicating a substantial reduction of human-vector contact shortly after nets implementation. Overall, children IgG response to the cE5 protein appeared a very sensitive biomarker, which allowed for the detection of even weak exposure to Anopheles bites, indicating it may represent a reliable additional tool to evaluate the efficacy of vector control interventions.

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.

Objective Because sex hormones may be involved in tumor initiation and progression, we analyzed the presence of hormone receptors in the transformation zone of the uterine cervix where the majority of human papillomavirus infections and associated (pre)neoplastic lesions develop. Study design By using 23 total hysterectomy samples from young women who underwent surgery for noncervical benign uterine disease, we analyzed, by immunohistologic techniques, the in situ expression of estrogen (E2-R) and progesterone (P4-R) receptors in the transformation zone and ectocervix of the same women. Results The expression of estrogen receptors and progesterone receptors is significantly higher in the transformation zone compared with the ectocervix. Immunohistochemical localization indicated that hormone receptor-positive cells are mainly observed in (para)basal and intermediate cell layers in both the transformation zone and ectocervical epithelium. When transformation zone samples were segregated into epithelial tissues with a predominantly mature (7/23 samples) or immature (16/23 samples) squamous metaplasia, only biopsy specimens with immature squamous metaplasia showed a significantly higher density of hormone receptor-positive cells compared with ectocervical epithelium (P Conclusion Our results suggest that the cervical transformation zone may be at increased risk of the development of cancer because of a high sensitivity to sex hormone regulation.

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.

Sex hormones seem to have an influence on the level of parasitic infection. Indeed, gender-dependent patterns of prevalence and intensity of infection after puberty have been observed for several parasite species (5). It has been suggested that this effect seems to be associated with the regulatory roles of sex steroids on antiparasite immunity (2, 24). Generally, female hormones have an influence in increasing antibody response against specific antigen, which could explain the higher resistance of women against several parasitic infections (24, 25). During human Schistosoma infection, a chronic infection affecting 200 million individuals around the world, sex hormones and particularly the high level of testosterone after puberty could be an important immune modulator leading to the decrease of susceptibility to infection with age (10, 26). In mice infected by Schistosoma mansoni, fewer adult worms develop in males than in similarly infected females (9). In addition, female mice treated with testosterone before infection presented a reduction in worm burden, whereas no difference in antischistosome immune response was detected between treated and untreated animals (20). These authors suggest thus that effects of testosterone on specific immunity are not adequate to explain the differences in parasite loads observed between the sexes. Schistosomes could express a hormone receptor with homology to the testosterone receptor, which could explain the direct effect of testosterone on worm development (8). Interestingly, it has been demonstrated that testosterone treatment can affect not only the development of Nippostrongylus brasiliensis helminthic worms but also the fecundity and maturity of laid eggs (27). Glutathione S-transferases (GSTs), a family of enzymes, are able to detoxify electrophilic compounds by catalyzing the formation of glutathione conjugates (12). Mammal GSTs are also involved in the intracellular transport of a variety of endogenous metabolites, drugs, and hormones by their abilities to bind these substances (16). Particularly, GSTs are glucocorticoid-binding proteins and, thereby, may influence transport, metabolism, and action of steroids (13). It has also been demonstrated that testosterone and progesterone have the ability to bind mammal GSTs with moderate (10−6 < Kd < 10−4 M) or high (Kd < 10−6 M) affinity, respectively (16). Thus, GSTs are also involved in the transport of sexual steroids and could play a key role in the physiological action of these hormones. The Schistosoma 28-kDa GST (28GST) is an essential enzyme for the parasite life in its host and is now a vaccine candidate against schistosomiasis (6). Immunization with recombinant 28GST Schistosoma haematobium (Sh28GST) has been shown to reduce S. haematobium fecundity in experimentally infected monkeys (4). It is well established in rodents (30) and observed during human infection (11) that this antifecundity effect is associated with the inhibition of the 28GST enzymatic activity by recognation of specific antibodies. Particularly, antibodies directed against amino acids 24 to 43 or 190 to 211 involved in the enzymatic site of the 28GST inhibited the GST activity, reducing tissue egg number and egg viability (31). Ultrastructural localization of antigen in adult worms showed that 28GST was identified in the tegument and the parenchyma, but also in the germinal organ of both male and female parasites (17, 21). These results indicate that 28GST expression seems to be closely associated with parasite metabolism but also with the genital tract, which could explain the relationship between the inhibition of enzymatic activity and an antifecundity effect. The aim of our study was to demonstrate the potential binding of testosterone to Sh28GST and evaluate the functional ability of this binding on the enzymatic activity of the parasite protein.

To evaluate immunity to vaccine-preventable diseases according to nutritional status, a longitudinal study was conducted in Senegalese children ages 1-9 years old. A linear regression analysis predicted that weight for age was positively associated with immunoglobulin G (IgG) response to tetanus toxoid in children born during the rainy season or at the beginning of the dry season. A relationship between village, time of visits, and levels of antibodies to tetanus showed that environmental factors played a role in modulating humoral immunity to tetanus vaccine over time. Moreover, a whole-blood stimulation assay highlighted that the production of interferon-gamma (IFN-gamma) in response to tetanus toxoid was compromised in stunted children. However, the absence of cytokine modulation in response to Mycobacterium tuberculosis-purified protein derivatives and phytohemagglutinin suggests that the overall ability to produce IFN-gamma was preserved in stunted children. Therefore, these results show that nutritional status can specifically alter the efficacy of long-lasting immunity to tetanus.

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.

Aedesborne viruses represent public health problems in southern countries and threat to emerge in the developed world. Their control is currently based on vector population control. Much effort is being devoted to develop new tools to control such arbovirus. Recent findings suggest that the evaluation of human antibody (Ab) response to arthropod salivary proteins is relevant to measuring the level of human exposure to mosquito bites. Using an immunoepidemiological approach, the present study aimed to assess the usefulness of the salivary biomarker for measuring the efficacy ofAe. albopictuscontrol strategies in La Reunion urban area. The antisaliva Ab response of adult humans exposed toAe. albopictuswas evaluatedbefore and after vector control measures. Our results showed a significant correlation between antisaliva Ab response and the level of exposure to vectors bites. The decrease ofAe.albopictusdensity has been detected by this biomarker two weeks after the implementation of control measures, suggesting its potential usefulness for evaluating control strategies in a short time period. The identification of species specific salivary proteins/peptides should improve the use of this biomarker.

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.

Background While vaccines elicit a protective response in most recipients, studies suggest that environmental and nutritional factors can influence the strength of the individual response to immunization and to subsequent natural infectious challenges. Methods We conducted a longitudinal survey in Senegal to assess the individual response to B. pertussis, a respiratory disease against which Senegalese children are vaccinated before the age of one (Clinicaltrials.gov ID: NCT01545115). A cohort of 203 children aged 1–9 from four villages of the Senegal River Valley was followed-up for 14 months (October 2008–January 2010). During that period, four visits have been made to the villages to assess the immunological and nutritional status of these children and to determine risk factors involved in the modulation of their humoral immune response to B. pertussis toxin. Results A multivariate model has demonstrated that birth season and nutritional status appeared to modulate humoral response to pertussis toxin. Moreover, response to B. pertussis was dependent on age, village and time of visit. Conclusions These results are consistent with the hypothesis that environmental and nutritional factors modulate children's response to pertussis following natural infection or vaccination.

A recently reported epidemic of Schistosoma mansoni infection in Senegal provided an opportunity to study the dynamics of the development of immunity to human schistosomiasis. We report here on the cell-mediated immune response in a population of 99 females and 95 males, with particular emphasis on the relationship between intensity of infection and age. We found that the intensity of infection correlated negatively with age in females but not in males. In men and women, both Th1- and Th2-type cytokines were detected upon in vitro stimulation of PBMCs with soluble egg antigen (SEA) or soluble adult worm antigens (SWAP). In the female group, SEA-induced PBMC proliferation was associated with the production of IFN-gamma, IL-2 and IL-5, all of which correlated negatively with intensity of infection. Most cytokine production correlated positively with age. Spontaneous production of TNF-alpha, IL-6 and IL-10 was higher in the infected population than in an uninfected control group. Our results suggest that immunity to infection could be more pronounced in the female population and associated with a Th0/1 + 2 pattern of cytokine secretion mediated by soluble egg antigen (SEA).

For the development of vaccine strategies to generate efficient protection against chronic infections such as parasitic diseases, and more precisely schistosomiasis, controlling pathology could be more relevant than controlling the infection itself. Such strategies, motivated by the need for a cost-effective complement to existing control measures, should focus on parasite molecules involved in fecundity, because in metazoan parasite infections pathology is usually linked to the output of viable eggs. In numerous animal models, vaccination with glutathione S-transferases of 28kDa has been shown to generate an immune response strongly limiting the worm fecundity, in addition to the reduction of the parasite burden. Recent data on acquired immunity directed to 28GST in infected human populations, and new development to draw adapted vaccine formulations, are presented.

Inflammatory cytokines have been described to play a critical role in the orientation and amplification of the IgA immune response. In this study, we show that the intranasal administration of a Bordetella pertussis strain expressing the protective antigen glutathione-S-transferase of Schistosoma mansoni (Sm28GST) induced an inflammatory response in the lungs of mice, characterized by the production of inflammatory cytokines, such as Tumor Necrosis Factor alpha, Interleukin-6 and Transforming-Growth Factor beta. The production and the secretion of these cytokines in lung tissues were early and transient. Their presence was observed only during the first week after administration despite the persistence of the bacteria for 1 month. Two weeks after inoculation, Interleukin-10 secretion was detected in the lungs, which could explain the decrease in the production of inflammatory cytokines. These inflammation-regulating cytokines, induced in the lungs by the presence of the bacterial vector, could be part of the process generating the local immune response, in particular the anti-Sm28GST IgA response.

International audience

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.