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Abstract Many factors are involved in determining the latitudinal and altitudinal spread of the important tick vector Ixodes ricinus (Acari: Ixodidae) in Europe, as well as in changes in the distribution within its prior endemic zones. This paper builds on published literature and unpublished expert opinion from the VBORNET network with the aim of reviewing the evidence for these changes in Europe and discusses the many climatic, ecological, landscape and anthropogenic drivers. These can be divided into those directly related to climatic change, contributing to an expansion in the tick’s geographic range at extremes of altitude in central Europe, and at extremes of latitude in Scandinavia; those related to changes in the distribution of tick hosts, particularly roe deer and other cervids; other ecological changes such as habitat connectivity and changes in land management; and finally, anthropogenically induced changes. These factors are strongly interlinked and often not well quantified. Although a change in climate plays an important role in certain geographic regions, for much of Europe it is non-climatic factors that are becoming increasingly important. How we manage habitats on a landscape scale, and the changes in the distribution and abundance of tick hosts are important considerations during our assessment and management of the public health risks associated with ticks and tick-borne disease issues in 21st century Europe. Better understanding and mapping of the spread of I. ricinus (and changes in its abundance) is, however, essential to assess the risk of the spread of infections transmitted by this vector species. Enhanced tick surveillance with harmonized approaches for comparison of data enabling the follow-up of trends at EU level will improve the messages on risk related to tick-borne diseases to policy makers, other stake holders and to the general public.

Abstract Vector borne disease (VBD) emergence is a complex and dynamic process. Interactions between multiple disciplines and responsible health and environmental authorities are often needed for an effective early warning, surveillance and control of vectors and the diseases they transmit. To fully appreciate this complexity, integrated knowledge about the human and the vector population is desirable. In the current paper, important parameters and terms of both public health and medical entomology are defined in order to establish a common language that facilitates collaboration between the two disciplines. Special focus is put on the different VBD contexts with respect to the current presence or absence of the disease, the pathogen and the vector in a given location. Depending on the context, whether a VBD is endemic or not, surveillance activities are required to assess disease burden or threat, respectively. Following a decision for action, surveillance activities continue to assess trends.

Abstract Background The entomological inoculation rate (EIR) is an important indicator in estimating malaria transmission and the impact of vector control. To assess the EIR, the enzyme-linked immunosorbent assay (ELISA) to detect the circumsporozoite protein (CSP) is increasingly used. However, several studies have reported false positive results in this ELISA. The false positive results could lead to an overestimation of the EIR. The aim of present study was to estimate the level of false positivity among different anopheline species in Cambodia and Vietnam and to check for the presence of other parasites that might interact with the anti-CSP monoclonal antibodies. Methods Mosquitoes collected in Cambodia and Vietnam were identified and tested for the presence of sporozoites in head and thorax by using CSP-ELISA. ELISA positive samples were confirmed by a Plasmodium specific PCR. False positive mosquitoes were checked by PCR for the presence of parasites belonging to the Haemosporidia, Trypanosomatidae, Piroplasmida, and Haemogregarines. The heat-stability and the presence of the cross-reacting antigen in the abdomen of the mosquitoes were also checked. Results Specimens (N = 16,160) of seven anopheline species were tested by CSP-ELISA for Plasmodium falciparum and Plasmodium vivax (Pv210 and Pv247). Two new vector species were identified for the region: Anopheles pampanai (P. vivax) and Anopheles barbirostris (Plasmodium malariae). In 88% (155/176) of the mosquitoes found positive with the P. falciparum CSP-ELISA, the presence of Plasmodium sporozoites could not be confirmed by PCR. This percentage was much lower (28% or 5/18) for P. vivax CSP-ELISAs. False positive CSP-ELISA results were associated with zoophilic mosquito species. None of the targeted parasites could be detected in these CSP-ELISA false positive mosquitoes. The ELISA reacting antigen of P. falciparum was heat-stable in CSP-ELISA true positive specimens, but not in the false positives. The heat-unstable cross-reacting antigen is mainly present in head and thorax and almost absent in the abdomens (4 out of 147) of the false positive specimens. Conclusion The CSP-ELISA can considerably overestimate the EIR, particularly for P. falciparum and for zoophilic species. The heat-unstable cross-reacting antigen in false positives remains unknown. Therefore it is highly recommended to confirm all positive CSP-ELISA results, either by re-analysing the heated ELISA lysate (100°C, 10 min), or by performing Plasmodium specific PCR followed if possible by sequencing of the amplicons for Plasmodium species determination.

Abstract Background In Vietnam, malaria is becoming progressively restricted to specific foci where human and vector characteristics alter the known malaria epidemiology, urging for alternative or adapted control strategies. Long-lasting insecticidal hammocks (LLIH) were designed and introduced in Ninh Thuan province, south-central Vietnam, to control malaria in the specific context of forest malaria. An entomological study in this specific forested environment was conducted to assess the behavioural patterns of forest and village vectors and to assess the spatio-temporal risk factors of malaria transmission in the province. Methods Five entomological surveys were conducted in three villages in Ma Noi commune and in five villages in Phuoc Binh commune in Ninh Thuan Province, south-central Vietnam. Collections were made inside the village, at the plot near the slash-and-burn fields in the forest and on the way to the forest. All collected mosquito species were subjected to enzyme-linked immunosorbent assay (ELISA) to detect Plasmodium in the head-thoracic portion of individual mosquitoes after morphological identification. Collection data were analysed by use of correspondence and multivariate analyses. Results The mosquito density in the study area was low with on average 3.7 anopheline bites per man-night and 17.4 culicine bites per man-night. Plasmodium-infected mosquitoes were only found in the forest and on the way to the forest. Malaria transmission in the forested malaria foci was spread over the entire night, from dusk to dawn, but was most intense in the early evening as nine of the 13 Plasmodium positive bites occurred before 21H. The annual entomological inoculation rate of Plasmodium falciparum was 2.2 infective bites per person-year to which Anopheles dirus s.s. and Anopheles minimus s.s. contributed. The Plasmodium vivax annual entomological inoculation rate was 2.5 infective bites per person-year with Anopheles sawadwongporni, Anopheles dirus s.s. and Anopheles pampanai as vectors. Conclusion The vector behaviour and spatio-temporal patterns of malaria transmission in Southeast Asia impose new challenges when changing objectives from control to elimination of malaria and make it necessary to focus not only on the known main vector species. Moreover, effective tools to prevent malaria transmission in the early evening and in the early morning, when the treated bed net cannot be used, need to be developed.

Abstract Background In the Mekong region (Vietnam, Cambodia and Laos), a large investigation was conducted to assess the susceptibility of Anopheles species against DDT and pyrethroids. In this study, the resistance status of the potential malaria vectors An. vagus, An. sinensis, An. paraliae and An. peditaeniatus was assessed. Methods Bioassays were performed on field collected unfed female mosquitoes using the standard WHO susceptibility tests. In addition, the DIIS6 region of the para-type sodium channel gene was amplified and sequenced and four allele-specific PCR assays were developed to assess the kdr frequencies. Results In Southern Vietnam all species were DDT and pyrethroid resistant, which might suggest the presence of a kdr resistance mechanism. Sequence-analysis of the DIIS6 region of the para-type sodium channel gene revealed the presence of a L1014S kdr mutation in An. vagus, An. sinensis and An. paraliae. In An. peditaeniatus, a low frequency L1014S kdr mutation was found in combination with a high frequency L1014F kdr mutation. For pyrethroids and DDT, no genotypic differentiation was found between survivors and non-survivors for any of these species. In the two widespread species, An. vagus and An. sinensis, kdr was found only in southern Vietnam and in Cambodia near the Vietnamese border. Conclusions Different levels of resistance were measured in Laos, Cambodia and Vietnam. The kdr mutation in different Anopheles species seems to occur in the same geographical area. These species breed in open agricultural lands where malaria endemicity is low or absent and vector control programs less intensive. It is therefore likely that the selection pressure occurred on the larval stages by insecticides used for agricultural purposes.

Abstract Background Malaria remains the most important public health problem in tropical and subtropical areas. Mothers' or caregivers' ability to recognize childhood malaria-related morbidity is crucial as knowledge, attitudes and health seeking behavior of caregivers towards childhood malaria could influence response to signs of the disease. Methods A total of 1,003 caregivers in 'at-risk' villages in close proximity to the Gilgel-Gibe hydroelectric dam in south-western Ethiopia, and 953 caregivers in 'control' villages further away from the dam were surveyed using structured questionnaires to assess their knowledge, perceptions and health seeking behaviour about childhood malaria. Results Malaria (busa) was ranked as the most serious health problem. Caregivers perceived childhood malaria as a preventable ('at-risk' 96%, 'control' 86%) and treatable ('at-risk' 98% and 'control' 96%) disease. Most caregivers correctly associated the typical clinical manifestations with malaria attacks. The use of insecticide-treated nets (ITNs) was mentioned as a personal protective measure, whereas the role of indoor residual spraying (IRS) in malaria prevention and control was under-recognized. Most of the caregivers would prefer to seek treatment in health-care services in the event of malaria and reported the use of recommended anti-malarials. Conclusion Health education to improve knowledge, perceptions and health-seeking behaviour related to malaria is equally important for caregivers in 'at risk' villages and caregivers in 'control' villages as minimal differences seen between both groups. Concluding, there may be a need of more than one generation after the introduction of the dam before differences can be noticed. Secondly, differences in prevalence between 'control' and 'at-risk' villages may not be sufficient to influence knowledge and behaviour.

Abstract Background Despite Vietnam's success in reducing malaria mortality and morbidity over the last decade, malaria persists in the forested and mountainous areas of the central and southern provinces, where more than 50% of the clinical cases and 90% of severe cases and malaria deaths occur. Methods Between July 2005 and September 2006, a multi-method study, triangulating a malariometric cross-sectional survey and qualitative data from focused ethnography, was carried out among the Ra-glai ethnic minority in the hilly forested areas of south-central Vietnam. Results Despite the relatively high malaria burden among the Ra-glai and their general awareness that mosquitoes can transmit an unspecific kind of fever (84.2%), the use of bed nets, distributed free of charge by the national malaria control programme, remains low at the farmers' forest fields where the malaria risk is the highest. However, to meet work requirements during the labour intensive malaria transmission and rainy season, Ra-glai farmers combine living in government supported villages along the road with a second home or shelter at their slash and burn fields located in the forest. Bed net use was 84.6% in the villages but only 52.9% at the forest fields; 20.6% of the respondents slept unprotected in both places. Such low use may be explained by the low perception of the risk for malaria, decreasing the perceived need to sleep protected. Several reasons may account for this: (1) only 15.6% acknowledged the higher risk of contracting malaria in the forest than in the village; (2) perceived mosquito biting times only partially coincided with Anopheles dirus ss and Anopheles minimus A true biting times; (3) the disease locally identified as 'malaria' was hardly perceived as having an impact on forest farmers' daily lives as they were unaware of the specific kind of fevers from which they had suffered even after being diagnosed with malaria at the health centre (20.9%). Conclusions The progressive confinement of malaria to minority groups and settings in the Greater Mekong sub-region implies that further success in malaria control will be linked to research into these specific socio-cultural contexts. Findings highlight the need for context sensitive malaria control policies; not only to reduce the local malaria burden but also to minimize the risk of malaria spreading to other areas where transmission has virtually ceased.

Abstract Background In this study, the efficacy of insecticide-treated nets was evaluated in terms of deterrence, blood-feeding inhibition, induced exophily and mortality on a wild resistant population of Anopheles epiroticus in southern Vietnam, in order to gain insight into the operational consequences of the insecticide resistance observed in this malaria vector in the Mekong delta. Method An experimental station, based on the model of West Africa and adapted to the behaviour of the target species, was built in southern Vietnam. The study design was adapted from the WHO phase 2 guidelines. The study arms included a conventionally treated polyester net (CTN) with deltamethrin washed just before exhaustion, the WHO recommended long-lasting insecticidal net (LLIN) PermaNet 2.0® unwashed and 20 times washed and PermaNet 3.0®, designed for the control of pyrethroid resistant vectors, unwashed and 20 times washed. Results The nets still provided personal protection against the resistant An. epiroticus population. The personal protection ranged from 67% for deltamethrin CTN to 85% for unwashed PermaNet 3.0. Insecticide resistance in the An. epiroticus mosquitoes did not seem to alter the deterrent effect of pyrethroids. A significant higher mortality was still observed among the treatment arms despite the fact that the An. epiroticus population is resistant against the tested insecticides. Conclusion This study shows that CTN and LLINs still protect individuals against a pyrethroid resistant malaria vector from the Mekong region, where insecticide resistance is caused by a metabolic mechanism. In the light of a possible elimination of malaria from the Mekong region these insights in operational consequences of the insecticide resistance on control tools is of upmost importance.

Abstract Background As insecticide resistance may jeopardize the successful malaria control programmes in the Mekong region, a large investigation was previously conducted in the Mekong countries to assess the susceptibility of the main malaria vectors against DDT and pyrethroid insecticides. It showed that the main vector, Anopheles epiroticus, was highly pyrethroid-resistant in the Mekong delta, whereas Anopheles minimus sensu lato was pyrethroid-resistant in northern Vietnam. Anopheles dirus sensu stricto showed possible resistance to type II pyrethroids in central Vietnam. Anopheles subpictus was DDT- and pyrethroid-resistant in the Mekong Delta. The present study intends to explore the resistance mechanisms involved. Methods By use of molecular assays and biochemical assays the presence of the two major insecticide resistance mechanisms, knockdown and metabolic resistance, were assessed in the main malaria vectors of the Mekong region. Results Two FRET/MCA assays and one PCR-RFLP were developed to screen a large number of Anopheles populations from the Mekong region for the presence of knockdown resistance (kdr), but no kdr mutation was observed in any of the study species. Biochemical assays suggest an esterase mediated pyrethroid detoxification in An. epiroticus and An. subpictus of the Mekong delta. The DDT resistance in An. subpictus might be conferred to a high GST activity. The pyrethroid resistance in An. minimus s.l. is possibly associated with increased detoxification by esterases and P450 monooxygenases. Conclusion As different metabolic enzyme systems might be responsible for the pyrethroid and DDT resistance in the main vectors, each species may have a different response to alternative insecticides, which might complicate the malaria vector control in the Mekong region.

Abstract Background Ethiopia plans to increase its electricity power supply by five-fold over the next five years to fulfill the needs of its people and support the economic growth based on large hydropower dams. Building large dams for hydropower generation may increase the transmission of malaria since they transform ecosystems and create new vector breeding habitats. The aim of this study was to assess the effects of Gilgel-Gibe hydroelectric dam in Ethiopia on malaria transmission and changing levels of prevalence in children. Methods A cross-sectional, community-based study was carried out between October and December 2005 in Jimma Zone, south-western Ethiopia, among children under 10 years of age living in three 'at-risk' villages (within 3 km from dam) and three 'control' villages (5 to 8 km from dam). The man-made Gilgel-Gibe dam is operating since 2004. Households with children less than 10 years of age were selected and children from the selected households were sampled from all the six villages. This included 1,081 children from 'at-risk' villages and 774 children from 'control' villages. Blood samples collected from children using finger prick were examined microscopically to determine malaria prevalence, density of parasitaemia and identify malarial parasite species. Results Overall 1,855 children (905 girls and 950 boys) were surveyed. A total of 194 (10.5%) children were positive for malaria, of which, 117 (60.3%) for Plasmodium vivax, 76 (39.2%) for Plasmodium falciparum and one (0.5%) for both P. vivax and P. falciparum. A multivariate design-based analysis indicated that, while controlling for age, sex and time of data collection, children who resided in 'at-risk' villages close to the dam were more likely to have P. vivax infection than children who resided farther away (odds ratio (OR) = 1.63, 95% CI = 1.15, 2.32) and showed a higher OR to have P. falciparum infection than children who resided in 'control' villages, but this was not significant (OR = 2.40, 95% CI = 0.84, 6.88). A classification tree revealed insights in the importance of the dam as a risk factor for malaria. Assuming that the relationship between the dam and malaria is causal, 43% of the malaria occurring in children was due to living in close proximity to the dam. Conclusion This study indicates that children living in close proximity to a man-made reservoir in Ethiopia are at higher risk of malaria compared to those living farther away. It is recommended that sound prevention and control programme be designed and implemented around the reservoir to reduce the prevalence of malaria. In this respect, in localities near large dams, health impact assessment through periodic survey of potential vectors and periodic medical screening is warranted. Moreover, strategies to mitigate predicted negative health outcomes should be integral parts in the preparation, construction and operational phases of future water resource development and management projects.

Abstract Background Knowledge on insecticide resistance in target species is a basic requirement to guide insecticide use in malaria control programmes. Malaria transmission in the Mekong region is mainly concentrated in forested areas along the country borders, so that decisions on insecticide use should ideally be made at regional level. Consequently, cross-country monitoring of insecticide resistance is indispensable to acquire comparable baseline data on insecticide resistance. Methods A network for the monitoring of insecticide resistance, MALVECASIA, was set up in the Mekong region in order to assess the insecticide resistance status of the major malaria vectors in Cambodia, Laos, Thailand, and Vietnam. From 2003 till 2005, bioassays were performed on adult mosquitoes using the standard WHO susceptibility test with diagnostic concentrations of permethrin 0.75% and DDT 4%. Additional tests were done with pyrethroid insecticides applied by the different national malaria control programmes. Results Anopheles dirus s.s., the main vector in forested malaria foci, was susceptible to permethrin. However, in central Vietnam, it showed possible resistance to type II pyrethroids. In the Mekong delta, Anopheles epiroticus was highly resistant to all pyrethroid insecticides tested. It was susceptible to DDT, except near Ho Chi Minh City where it showed possible DDT resistance. In Vietnam, pyrethroid susceptible and tolerant Anopheles minimus s.l. populations were found, whereas An. minimus s.l. from Cambodia, Laos and Thailand were susceptible. Only two An. minimus s.l. populations showed DDT tolerance. Anopheles vagus was found resistant to DDT and to several pyrethroids in Vietnam and Cambodia. Conclusion This is the first large scale, cross-country survey of insecticide resistance in Anopheles species in the Mekong Region. A unique baseline data on insecticide resistance for the Mekong region is now available, which enables the follow-up of trends in susceptibility status in the region and which will serve as the basis for further resistance management. Large differences in insecticide resistance status were observed among species and countries. In Vietnam, insecticide resistance was mainly observed in low or transmission-free areas, hence an immediate change of malaria vector control strategy is not required. Though, resistance management is important because the risk of migration of mosquitoes carrying resistance genes from non-endemic to endemic areas. Moreover, trends in resistance status should be carefully monitored and the impact of existing vector control tools on resistant populations should be assessed.

Abstract Background The distribution of anopheline mosquitoes in Vietnam was examined, with a particular interest for the two sibling species of the Anopheles minimus complex (Cellia: Myzomyia), An. minimus and Anopheles harrisoni, respectively former species A and C. Because the morphological identification of both sibling species is difficult and may lead to misidentifications, accurate data on their respective distribution are missing. This is of fundamental importance since the two species seem to exhibit differential vectorial capacities for malaria transmission. Methods Large entomological surveys based on cattle collections and molecular identifications of An. minimus s.l. were carried out in 23 sites throughout northern, central and south-eastern regions of Vietnam. Results Based on previous molecular works and our data, the distribution of anopheline species and the relative densities of An. minimus and An. harrisoni were mapped. It is noteworthy that there was a high specific biodiversity at each study site. Anopheles minimus s.l. and Anopheles sinensis were the main anopheline species in the northern region, whereas Anopheles aconitus and Anopheles vagus were the most frequent ones in the central region. The southern limit of An. harrisoni was increased to the latitude of 11°N. Sympatry between both sibling species has been extended to new provinces. Conclusion Malaria transmission is still high in central Vietnam and along bordering countries. Therefore, it is important to know and map the precise distribution of the main and secondary malaria vectors in Vietnam for applying efficient vector control programmes. Moreover, these maps should be regularly updated and linked to environmental characteristics relative to disease epidemiology, and environmental and climatic changes occurring in southeast Asia.

Abstract Background Prevention of malaria epidemics is a priority for African countries. The 2000 malaria epidemic in Burundi prompted the government to implement measures for preventing future outbreaks. Case management with artemisinin-based combination therapy and malaria surveillance were nationally improved. A vector control programme was initiated in one of the most affected highland provinces. The focal distribution of malaria vectors in the highlands was the starting point for designing a targeted vector control strategy. The objective of this study was to present the results of this strategy on malaria transmission in an African highland region. Methods In Karuzi, in 2002–2005, vector control activities combining indoor residual spraying and long-lasting insecticidal nets were implemented. The interventions were done before the expected malaria transmission period and targeted the valleys between hills, with the expectation that this would also protect the populations living at higher altitudes. The impact on the Anopheles population and on malaria transmission was determined by nine cross-sectional surveys carried out at regular intervals throughout the study period. Results Anopheles gambiae s.l. and Anopheles funestus represented 95% of the collected anopheline species. In the valleys, where the vector control activities were implemented, Anopheles density was reduced by 82% (95% CI: 69–90). Similarly, transmission was decreased by 90% (95% CI: 63%–97%, p = 0.001). In the sprayed valleys, Anopheles density was further reduced by 79.5% (95% CI: 51.7–91.3, p < 0.001) in the houses with nets as compared to houses without them. No significant impact on vector density and malaria transmission was observed in the hill tops. However, the intervention focused on the high risk areas near the valley floor, where 93% of the vectors are found and 90% of the transmission occurs. Conclusion Spatial targeted vector control effectively reduced Anopheles density and transmission in this highland district. Bed nets have an additional effect on Anopheles density though this did not translate in an additional impact on transmission. Though no impact was observed in the hilltops, the programme successfully covered the areas most at risk. Such a targeted strategy could prevent the emergence and spread of an epidemic from these high risk foci.

Abstract Background African highlands often suffer of devastating malaria epidemics, sometimes in conjunction with complex emergencies, making their control even more difficult. In 2000, Burundian highlands experienced a large malaria outbreak at a time of civil unrest, constant insecurity and nutritional emergency. Because of suspected high resistance to the first and second line treatments, the provincial health authority and Médecins Sans Frontières (Belgium) decided to implement vector control activities in an attempt to curtail the epidemic. There are few reported interventions of this type to control malaria epidemics in complex emergency contexts. Here, decisions and actions taken to control this epidemic, their impact and the lessons learned from this experience are reported. Case description Twenty nine hills (administrative areas) were selected in collaboration with the provincial health authorities for the vector control interventions combining indoor residual spraying with deltamethrin and insecticide-treated nets. Impact was evaluated by entomological and parasitological surveys. Almost all houses (99%) were sprayed and nets use varied between 48% and 63%. Anopheles indoor resting density was significantly lower in treated as compared to untreated hills, the latter taken as controls. Despite this impact on the vector, malaria prevalence was not significantly lower in treated hills except for people sleeping under a net. Discussion Indoor spraying was feasible and resulted in high coverage despite being a logistically complex intervention in the Burundian context (scattered houses and emergency situation). However, it had little impact on the prevalence of malaria infection, possibly because it was implemented after the epidemic's peak. Nevertheless, after this outbreak the Ministry of Health improved the surveillance system, changed its policy with introduction of effective drugs and implementation of vector control to prevent new malaria epidemics. Conclusion In the absence of effective drugs and sufficient preparedness, present study failed to demonstrate any impact of vector control activities upon the course of a short-duration malaria epidemic. However, the experience gained lead to increased preparedness and demonstrated the feasibility of vector control measures in this specific context.

Abstract Background Appropriate monitoring of vector resistance to insecticides is an integral component of planning and evaluation of insecticide use in malaria control programmes. The malaria vectors Anopheles gambiae s.s. and Anopheles arabiensis have developed resistance to pyrethroid insecticides as a result of a mechanism conferring reduced nervous system sensitivity, better known as knockdown resistance (kdr). In An. gambiae s.s. and An. arabiensis, two different substitutions in the para-type sodium channel, a L1014F substitution common in West Africa and a L1014S replacement found in Kenya, are linked with kdr. Two different allele-specific polymerase chain reactions (AS-PCR) are needed to detect these known kdr mutations. However, these AS-PCR assays rely on a single nucleotide polymorphism mismatch, which can result in unreliable results. Methods Here, a new assay for the detection of knockdown resistance in An. gambiae s.s. and An. arabiensis based on Fluorescence Resonance Energy Transfer/Melt Curve analysis (FRET/MCA) is presented and compared with the existing assays. Results The new FRET/MCA method has the important advantage of detecting both kdr alleles in one assay. Moreover, results show that the FRET/MCA is more reliable and more sensitive than the existing AS-PCR assays and is able to detect new genotypes. By using this technique, the presence of the East African kdr mutation (L1014S) is shown for the first time in An. arabiensis specimens from Uganda. In addition, a new kdr genotype is reported in An. gambiae s.s. from Uganda, where four An. gambiae s.s. mosquitoes possess both, the West (L1014F) and East (L1014S) African kdr allele, simultaneously. Conclusion The presence of both kdr mutations in the same geographical region shows the necessity of a reliable assay that enables to detect both mutations in one single assay. Hence, this new assay based on FRET/MCA will improve the screening of the kdr frequencies in An. gambiae s.s. and An. arabiensis.