Reconsideration of importance of the point mutation L982W in the voltage-sensitive sodium channel of the pyrethroid resistant Aedes aegypti (L.)(Diptera: Culicidae) in Vietnam.

Pyrethroid resistance in Aedes aegypti is widespread in southern Vietnam because the photostable 2nd generation pyrethroids have been used in large amounts over extensive areas for malaria and dengue vector control. In our previous report in 2009, F1534C, one of the point mutations in the voltage-sensitive sodium channel (VSSC) in Ae. aegypti, was widespread at high frequency in south and central area. However, no significant correlation between the frequency of F1534C and pyrethroid susceptibility was detected primarily because the F1534C mutation frequency in the southern highland area was very low, despite that the bioassay indicated high pyrethroid resistance. The point mutation in the VSSC, L982W, which was not the target mutation in our previous study, was recently determined to be an important mutation causing high-pyrethroid resistance in Vietnamese Ae. aegypti. In the present study, a re-investigation of L982W in the mosquito samples collected in 2006-2008 revealed a greater distribution of this mutation (allelic percentage 59.2%) than F1534C (21.7%) and the greater proportion of homozygous L982W as compared to F1534C provided a plausible answer to the question concerning the unknown resistance factor in the southern highland area. L982W frequencies were uniformly higher in the southern part of Vietnam, including the highland area with a significantly high positive correlation with pyrethroid resistance in Ae. aegypti.

Discovery of super-insecticide-resistant dengue mosquitoes in Asia: Threats of concomitant knockdown resistance mutations.

Aedes aegypti (Linnaeus, 1762) is the main mosquito vector for dengue and other arboviral infectious diseases. Control of this important vector highly relies on the use of insecticides, especially pyrethroids. The high frequency (>78%) of the L982W substitution was detected at the target site of the pyrethroid insecticide, the voltage-gated sodium channel (Vgsc) of A. aegypti collected from Vietnam and Cambodia. Alleles having concomitant mutations L982W + F1534C and V1016G + F1534C were also confirmed in both countries, and their frequency was high (>90%) in Phnom Penh, Cambodia. Strains having these alleles exhibited substantially higher levels of pyrethroid resistance than any other field population ever reported. The L982W substitution has never been detected in any country of the Indochina Peninsula except Vietnam and Cambodia, but it may be spreading to other areas of Asia, which can cause an unprecedentedly serious threat to the control of dengue fever as well as other Aedes-borne infectious diseases.

Effects of Indoor Environmental Factors and House Structures on Vaporization of Active Ingredient from Spatial Repellent Devices in Rural Houses in Malawi.

The use of a metofluthrin-impregnated spatial repellent device (MSRD) is a new and effective method for preventing mosquito blood feeding. Indoor environmental factors such as room temperature and ventilation rate are thought to be important for MSRD activity. Measurements of room temperature and vaporization of metofluthrin from MSRD in typical rural metal-roof and thatched-roof houses in southeastern Malawi were conducted. The relationship between house structure and the number of collected Anopheline mosquitoes with and without MSRD treatment was also investigated. The difference between daytime and nighttime room temperature was significantly higher in metal-roof houses than in thatched-roof houses. The vaporization of metofluthrin from the MSRD was not accelerated by the high room temperature, but by the high indoor air flow by ventilation. The number of mosquito collections was significantly higher in thatched-roof houses than in metal-roof houses. MSRD-treated thatched-roof houses have a higher probability of mosquito infestation, but the vaporization of metofluthrin is also higher because of indoor air flow, resulting in a reduction in mosquito numbers. Metal-roof houses with closed eaves reduce the probability of mosquito invasion, and a longer predicted effectiveness occurs with MSRD because of the controlled release of metofluthrin through lower indoor air flow.

Searching for a sign of exotic Aedes albopictus (Culicidae) introduction in major international seaports on Kyushu Island, Japan.

BACKGROUND: The Asian tiger mosquito, Aedes albopictus, has spread around the world. The migration was mainly mediated by maritime transportations. This species is known as an efficient vector for arboviruses, and it was responsible for the recent dengue outbreak in Tokyo, Japan. As the vector competence varies among geographical populations, and insecticide resistant populations have emerged, it is important to reveal their movements. The present study uses molecular techniques to search for a sign of introduction of an exotic population in three major international seaports on Kyushu Island. METHODOLOGY/PRINCIPAL FINDINGS: Adults of Ae. albopictus were sampled around the international seaports of Fukuoka, Kitakyushu, and Nagasaki. Pairwise fixation indexes were estimated between the sampled populations based on 13 microsatellite markers. There was no clear genetic differentiation between distant and port populations in Kitakyushu and Nagasaki. However, the analysis found one distinct group near the container terminal in Fukuoka, which handles international freight containers mainly from adjacent countries. DNA samples were also obtained from Goto, Tsushima, Honshu, Ryukyu, Thailand, and the Philippines; and a cluster analysis and discriminant analysis revealed that the distinct group in Fukuoka did not belong to these groups. Combined with the results of phylogenetic analysis based on CO1, these results implied that this group originated from one Asian temperate region outside of Japan. Neutrality test and mismatch distribution analysis suggested that the establishment of this group was not recent. CONCLUSIONS/SIGNIFICANCE: The present study found a sign of Ae. albopictus introduction from a temperate region of Asia through maritime freight container transportation. The genetically distinct group found in Fukuoka likely originated from a temperate region outside of Japan. Maritime container transportation may introduce to Japan mosquitoes with greater vector competence/insecticide resistance. This is the first study to describe the spatial population structure of Ae. albopictus in Japan using molecular techniques.

Long-Lasting Insecticidal Nets Incorporating Piperonyl Butoxide Reduce the Risk of Malaria in Children in Western Kenya: A Cluster Randomized Controlled Trial.

Malaria vectors have acquired an enzyme that metabolizes pyrethroids. To tackle this problem, we evaluated long-lasting insecticidal nets incorporating piperonyl butoxide (PBO-LLINs) with a community-based cluster randomized control trial in western Kenya. The primary endpoints were anopheline density and Plasmodium falciparum polymerase chain reaction (PCR)-positive prevalence (PCRpfPR) of children aged 7 months to 10 years. Four clusters were randomly selected for each of the treatment and control arms (eight clusters in total) from 12 clusters, and PBO-LLINs and standard LLINs were distributed in February 2011 to 982 and 1,028 houses for treatment and control arms, respectively. Entomological surveys targeted 20 houses in each cluster, and epidemiological surveys targeted 150 children. Cluster-level permutation tests evaluated the effectiveness using the fitted values from individual level regression models adjusted for baseline. Bootstrapping estimated 95% confidence intervals (CIs). The medians of anophelines per house were 1.4 (interquartile range [IQR]: 2.3) and 3.4 (IQR: 3.7) in the intervention and control arms after 3 months, and 0.4 (IQR: 0.2) and 1.6 (IQR: 0.5) after 10 months, respectively. The differences were -2.5 (95% CI: -6.4 to -0.6) and -1.3 (95% CI: -2.0 to -0.7), respectively. The datasets of 861 and 775 children were analyzed in two epidemiological surveys. The median PCRpfPRs were 25% (IQR: 11%) in the intervention arm and 52% (IQR: 11%) in the control arm after 5 months and 33% (IQR: 11%) and 45% (IQR: 5%) after 12 months. The PCRpfPR ratios were 0.67 (95% CI: 0.38, 0.91) and 0.74 (95% CI: 0.53, 0.90), respectively. We confirmed the superiority of PBO-LLINs.

Efficacy and safety of a dimethicone lotion in patients with pyrethroid-resistant head lice in an epidemic area, Okinawa, Japan.

Pyrethroid insecticides are the only pesticides approved for the treatment of head lice (pediculosis capitis) infestations in Japan. However, in Okinawa Prefecture, 96% of head lice are resistant to pyrethroids. Here, we conducted a clinical trial to assess the safety and efficacy of a dimethicone preparation against head lice infestations in Okinawa Prefecture. Dimethicone-containing lotion was applied over the entire scalp three times over a 7-day period. Lice bodies (nymphs/adults) and eggs (nits) were counted before (day 0) and after treatment (day 8); a subset of eggs was collected to estimate viability based on hatch rate. Efficacy was evaluated based on improvement (reduction) in head lice counts post-treatment with respect to baseline. Safety was evaluated based on subjects' scalp condition and adverse event incidence. Utility was a composite end-point combining efficacy and safety. In total, 35 subjects were enrolled. Efficacy and safety were evaluated in 23 and 35 subjects, respectively. No side-effects of note were reported during the treatment period. The dimethicone lotion resulted in a utility rating of "marginally useful" or higher in over 80% of the study population, signifying the formulation to be both safe and effective. The dimethicone lotion was also a potent ovicide; 99.4% of eggs collected after treatment failed to hatch when incubated. Eradication of head lice remained successful for at least 4 weeks after the final topical dimethicone application in 25 of the 28 subjects reached by telephone survey. Lice bodies and eggs were genotyped to analyze the prevalence of three knockdown resistance (kdr)-type mutations within the voltage-sensitive sodium channel known to confer pyrethroid resistance. One or more kdr mutations were confirmed in 30 of the 32 subjects from whom specimens were collected (93.8%). Dimethicone was confirmed to be both safe and effective in treating pyrethroid-resistant head lice.

New mosquito repellency bioassay for evaluation of repellents and pyrethroids using an attractive blood-feeding device.

BACKGROUND: With the increasing threat of the worldwide spread of mosquito-borne infectious diseases, consumer interest in anti-mosquito textiles that protect against mosquito bites is also increasing. Accordingly, repellent- or insecticide-treated textiles are gaining popularity. The standardization of commercial textile products is, therefore, indispensable for an authentic and objective evaluation of these products. Here we report a textile testing method using an artificial blood-feeding system that does not involve human volunteers or live animals, which aligns with the policy of protecting human and animal welfare. METHODS: The attractive blood-feeding device (ABFD) was designed using the Hemotek® membrane feeding system. The repellency of DEET, icaridin and permethrin was assayed using unfed female adults of Aedes albopictus (Skuse) under two different test conditions, namely choice and no-choice tests. The choice test consisted of two feeding units, one chemically treated and untreated, that were installed on the ABFD; mosquitoes attracted to and resting on the feeding units were counted and the overall blood-feeding rates recorded. The no-choice test consisted of two feeding units treated with the same chemical that were installed on the ABFD; mosquitoes attracted to and resting on the feeding units were counted and the blood-feeding rates were recorded. A control test was conducted using two feeding units, both sides of which were untreated. RESULTS: In the choice test, high repellency (> 95% inhibition of resting on the treated surface) of 1% DEET and 2% icaridin was observed, whereas 2% permethrin was not an effective repellent. Also, high blood-feeding inhibition (> 95%) was observed for 2% DEET and 2% icaridin. In the no-choice test, high repellency was observed for 1% DEET and 2% icaridin, whereas the repellency of 2% permethrin was low. Also, high blood-feeding inhibition was observed for 2% DEET, 4% icaridin and 2% permethrin. CONCLUSIONS: The accuracy and reproducibility of the developed method demonstrate that the ABFD may be widely used for fundamental experiments in the field of mosquito physiology, for the development of new repellent chemicals and in evaluation studies of mosquito repellent products, such as anti-mosquito textiles. The further development of the membrane and feeding unit systems will enable a more practical evaluation of mosquito repellents and blood-feeding inhibitors, such as pyrethroids.

A preliminary study on designing a cluster randomized control trial of two new mosquito nets to prevent malaria parasite infection.

BACKGROUND: Although long-lasting insecticidal nets (LLINs) are the most effective tool for preventing malaria parasite transmission, the nets have some limitations. For example, the increase of LLIN use has induced the rapid expansion of mosquito insecticide resistance. More than two persons often share one net, which increases the infection risk. To overcome these problems, two new mosquito nets were developed, one incorporating piperonyl butoxide and another covering ceilings and open eaves. We designed a cluster randomized controlled trial (cRCT) to evaluate these nets based on the information provided in the present preliminary study. RESULTS: Nearly 75% of the anopheline population in the study area in western Kenya was Anopheles gambiae s. l., and the remaining was Anopheles funestus s. l. More female anophelines were recorded in the western part of the study area. The number of anophelines increased with rainfall. We planned to have 80% power to detect a 50% reduction in female anophelines between the control group and each intervention group. The between-cluster coefficient of variance was 0.192. As the number of clusters was limited to 4 due to the size of the study area, the estimated cluster size was 7 spray catches with an alpha of 0.05. Of 1619 children tested, 626 (48%) were Plasmodium falciparum positive using a rapid diagnostic test (RDT). The prevalence was higher in the northwestern part of the study area. The number of children who slept under bed nets was 929 (71%). The P. falciparum RDT-positive prevalence (RDTpfPR) of net users was 45%, and that of non-users was 55% (OR 0.73; 95% CI 0.56, 0.95). Using 45% RDTpfPR of net users, we expected each intervention to reduce prevalence by 50%. The intracluster correlation coefficient was 0.053. With 80% power and an alpha of 0.05, the estimated cluster size was 116 children. Based on the distribution of children, we modified the boundaries of the clusters and established 300-m buffer zones along the boundaries to minimize a spillover effect. CONCLUSIONS: The cRCT study design is feasible. As the number of clusters is limited, we will apply a two-stage procedure with the baseline data to evaluate each intervention.

Distribution and pyrethroid resistance status of Aedes aegypti and Aedes albopictus populations and possible phylogenetic reasons for the recent invasion of Aedes aegypti in Nepal.

BACKGROUND: When the first systematic list of mosquitoes in Nepal was reported in 1990, there was no description of Aedes aegypti (L.), while Aedes albopictus (Skuse) has been included in the Stegomyia subgroup since the 1950s. The first record of Ae. aegypti in Nepal was reported in 2009, suggesting some coincidence between the invasion of this species and the first record of dengue fever in Nepal in 2006. RESULTS: We performed a field survey of the distribution and insecticide susceptibility of Ae. aegypti and Ae. albopictus in Nepal in 2017 and 2018. Mosquito larvae were collected from used tires located along the streets of Kathmandu, Bharatpur and Pokhara, and a simplified bioassay was used to assess the susceptibility of the larvae to pyrethroid insecticides using d-allethrin. The presence or absence of point mutations in the voltage-gated sodium channel was also detected by direct sequencing. V1016G was detected at a high frequency and a strong correlation was observed between the frequencies of V1016G and susceptibility indices in Ae. aegypti populations. F1534C was also detected at a relatively low frequency. In Ae. albopictus populations, susceptibilities to d-allethrin were high and no point mutations were detected. Analysis of the cytochrome c oxidase subunit 1 (cox1) gene was performed for assessing genetic diversity and the existence of two strains were identified in Ae. aegypti populations. One consisted of 9 globally-distributed haplotypes while the other was derived from an African haplotype. CONCLUSIONS: The high pyrethroid resistance, high V1016G frequency, and relatively low quantity of insecticides used to control dengue vectors in Nepal may have resulted in only weak selection pressure favoring insecticide resistance and could support the hypothesis that this species has recently been introduced from neighboring Asian countries where pyrethroid resistance is relatively widespread.

Effect of Metofluthrin-Impregnated Spatial Repellent Devices Combined with New Long-Lasting Insecticidal Nets (Olyset® Plus) on Pyrethroid-Resistant Malaria Vectors and Malaria Prevalence: Field Trial in South-Eastern Malawi.

Long-lasting insecticidal nets (LLINs) experience some operational problems that reduce their effectiveness, such as limited spaces for hanging, biting of mosquitoes outdoors, a shift of key biting time from midnight to dawn or dusk, and development of pyrethroid resistance in mosquitoes. The concept of spatial repellency may be a countermeasure to overcome the above issues. The effect of the combined use of metofluthrin-impregnated spatial repellent devices (MSRDs) and LLINs (Olyset® Plus) on malaria prevalence and vector mosquitoes were examined in malaria endemic villages in south-eastern Malawi. The intervention reduced the infection rate in children as well as the number of pyrethroid-resistant vector mosquitoes. To achieve effective malaria control, continued intervention using MSRDs with 2 strips per 10 m2 at 3-month intervals to reduce the density of malaria mosquitoes is recommended.

Analysis-ready datasets for insecticide resistance phenotype and genotype frequency in African malaria vectors.

The impact of insecticide resistance in malaria vectors is poorly understood and quantified. Here a series of geospatial datasets for insecticide resistance in malaria vectors are provided, so that trends in resistance in time and space can be quantified, and the impact of resistance found in wild populations on malaria transmission in Africa can be assessed. Specifically, data have been collated and geopositioned for the prevalence of insecticide resistance, as measured by standard bioassays, in representative samples of individual species or species complexes. Data are provided for the Anopheles gambiae species complex, the Anopheles funestus subgroup, and for nine individual vector species. Data are also given for common genetic markers of resistance to support analyses of whether these markers can improve the ability to monitor resistance in low resource settings. Allele frequencies for known resistance-associated markers in the Voltage-gated sodium channel (Vgsc) are provided. In total, eight analysis-ready, standardised, geopositioned datasets encompassing over 20,000 African mosquito collections between 1957 and 2017 are released.

Mode of entry of a vaporized pyrethroid knockdown agent into the body of the housefly, Musca domestica (Diptera: Muscidae).

We investigated the mode of entry of pyrethroids into the insect body using adult housefly, Musca domestica L., as an insect model. The wings of adult female houseflies were removed, and empenthrin was applied topically to three different sites: the mesothoracic spiracle, the ventral mesothorax, and the dorsal mesothorax. Among these treatments, the application of the compound to the mesothoracic spiracle led to the quickest knockdown of the flies. To determine the importance of the spiracle as a primary entry site for the pyrethroid, knockdown times were compared between houseflies with blocked and non-blocked spiracles, using two bioassays: a vapor action test using technical grade empenthrin, and a mosquito coil test using empenthrin-impregnated coils. In both tests, the times required for 50 % knockdown of spiracle-blocked houseflies were significantly higher than those required for the non-blocked flies. However, the mortality rates of the two groups were nearly identical, suggesting that spiracles play an important role in the knockdown of houseflies. These results also suggest that the rate of pyrethroid uptake through the spiracles was decreased due to the blocking of the mesothoracic spiracle. Therefore, the spiracle may be considered the main entry site for vaporized pyrethroids.

Discovery of Point Mutations in the Voltage-Gated Sodium Channel from African Aedes aegypti Populations: Potential Phylogenetic Reasons for Gene Introgression.

BACKGROUND: Yellow fever is endemic in some countries in Africa, and Aedes aegpyti is one of the most important vectors implicated in the outbreak. The mapping of the nation-wide distribution and the detection of insecticide resistance of vector mosquitoes will provide the beneficial information for forecasting of dengue and yellow fever outbreaks and effective control measures. METHODOLOGY/PRINCIPAL FINDINGS: High resistance to DDT was observed in all mosquito colonies collected in Ghana. The resistance and the possible existence of resistance or tolerance to permethrin were suspected in some colonies. High frequencies of point mutations at the voltage-gated sodium channel (F1534C) and one heterozygote of the other mutation (V1016I) were detected, and this is the first detection on the African continent. The frequency of F1534C allele and the ratio of F1534C homozygotes in Ae. aegypti aegypti (Aaa) were significantly higher than those in Ae. aegypti formosus (Aaf). We could detect the two types of introns between exon 20 and 21, and the F1534C mutations were strongly linked with one type of intron, which was commonly found in South East Asian and South and Central American countries, suggesting the possibility that this mutation was introduced from other continents or convergently selected after the introgression of Aaa genes from the above area. CONCLUSIONS/SIGNIFICANCE: The worldwide eradication programs in 1940s and 1950s might have caused high selection pressure on the mosquito populations and expanded the distribution of insecticide-resistant Ae. aegypti populations. Selection of the F1534C point mutation could be hypothesized to have taken place during this period. The selection of the resistant population of Ae. aegypti with the point mutation of F1534C, and the worldwide transportation of vector mosquitoes in accordance with human activity such as trading of used tires, might result in the widespread distribution of F1534C point mutation in tropical countries.

Isolation of proflavine as a blocker of G protein-gated inward rectifier potassium channels by a cell growth-based screening system.

The overexpression of Kir3.2, a subunit of the G protein-gated inwardly rectifying K(+) channel, is implicated in some of the neurological phenotypes of Down syndrome (DS). Chemical compounds that block Kir3.2 are expected to improve the symptoms of DS. The purpose of this study is to develop a cell-based screening system to identify Kir3.2 blockers and then investigate the mode of action of the blocker. Chemical screening was carried out using a K(+) transporter-deficient yeast strain that expressed a constitutively active Kir3.2 mutant. The mode of action of an effective blocker was electrophysiologically analyzed using Kir channels expressed in Xenopus oocytes. Proflavine was identified to inhibit the growth of Kir3.2-transformant cells and Kir3.2 activity in a concentration-dependent manner. The current inhibition was strong when membrane potentials (Vm) was above equilibrium potential of K(+) (EK). When Vm was below EK, the blockage apparently depended on the difference between Vm and [K(+)]. Furthermore, the inhibition became stronger by lowering extracellular [K(+)]. These results indicated that the yeast strain serves as a screening system to isolate Kir3.2 blockers and proflavine is a prototype of a pore blocker of Kir3.2.

Effect of Olyset Net on Mosquito Larvae and Their Heteroptera Predators in Water Jars in Southern Vietnam.

Residents of Vietnam living in areas with water shortages collect and store tap water, when it is available, in containers, such as jars, which is then used for their daily activities. Although these water jars are important breeding sites for mosquitoes, Vietnam legislation prohibits the use of chemical larvicides in such containers. The effect of Olyset Net on the abundance of mosquito larvae and their Heteroptera predators (Micronecta spp. and Veliidae) in jars was evaluated via field experiments conducted in Tan Chanh, Long An, Vietnam. The number of Aedes larvae decreased when the jars were covered with the Olyset Net, while the numbers of non-Aedes mosquito larvae (Culex and Anopheles), and those of Micronecta and Veliidae, did not vary until the end of the experiment. However, Micronecta population increased after removing the Olyset Net from the jars, and consequently Aedes larvae decreased due to these changes in the Micronecta, 26 days after the Olyset Net was removed. Incomplete use of Olyset Net failed to control mosquitoes in water jars based on the invasion of adult mosquitoes, and in water transferred by residents among the jars, which might contain larvae. These results suggest that Micronecta, as well as Olyset Net, can be considered as control agents for mosquito larvae.

Sleeping on the floor decreases insecticide treated bed net use and increases risk of malaria in children under 5 years of age in Mbita District, Kenya.

Children who sleep on the floor are less likely to use long-lasting insecticidal nets (LLINs); however, the relationship between sleeping location and Plasmodium falciparum infection has not been investigated sufficiently. This study revealed whether sleeping location (bed vs floor) is associated with P. falciparum infection among children 7-59 months old. More than 60% of children slept on the floor. Younger children were significantly more likely to sleep in beds [odds ratio, OR 2.31 (95% confidence interval (CI) 2.02-2.67)]. Nearly 70% of children slept under LLINs the previous night. LLIN use among children who slept on the floor was significantly less than ones sleeping in beds [OR 0.49 (95% CI 0.35-0.68)]. The polymerase chain reaction (PCR) based P. falciparum infection rate and slide based infection rate were 65.2 and 29.7%, respectively. Both infections were significantly higher among children slept on the floor [OR1.51 (95% CI 1.08-2.10) for PCR base, OR 1.62 (95% CI 1.14-2.30) for slide base] while net availability was not significant. Sleeping location was also significant for slide based infection with fever (⩾ 37.5 °C) [2.03 (95% CI 1.14-3.84)] and high parasitemia cases (parasite ⩾ 2500 µL(-1)) [2.07 (95% CI 1.03-4.50)]. The results suggest that sleeping location has a direct bearing on the effectiveness of LLINs.

A small-scale field trial of pyriproxyfen-impregnated bed nets against pyrethroid-resistant Anopheles gambiae s.s. in western Kenya.

Pyrethroid resistance is becoming a major problem for vector control programs, because at present, there are few suitable chemical substitutes for pyrethroids, as when used on bed nets the insecticide must have low mammalian toxicity as well as high activity to mosquitoes. Pyriproxyfen (PPF) is one of the most active chemicals among the juvenile hormone mimic (JHM) group. Sterilizing mosquitoes by using PPF could be a potential control measure for pyrethroid-resistant malaria vectors. We investigated the sterilizing effects of two types of PPF-impregnated bed nets - a 1% PPF-impregnated net and a 1% PPF +2% permethrin-impregnated net (Olyset Duo) - to pyrethroid-resistant wild population of Anopheles gambiae s.s. in western Kenya. High mortality of blood-fed mosquitos was observed 3 days post-collection, in the houses where PPF-impregnated nets were used, indicating the effect of PPF on the longevity of mosquitos that came in contact with the net. Reduction in the number of ovipositing females, number of eggs, and number of progeny per female were also observed in the houses in which both Olyset Duo and PPF-impregnated nets were used. This is the first field study showing the high sterilizing efficacy of PPF against wild pyrethroid-resistant An. gambiae s.s. population. In addition, we recognized the necessity of combined use of permethrin with PPF, in order to reduce the risk of mosquito bites and provide a level of personal protection. Further studies on wild pyrethroid-resistant mosquito populations such as An. arabiensis and An. funestus s.s. would provide more information on the practical use of the PPF-impregnated bed nets.

Co-occurrence of point mutations in the voltage-gated sodium channel of pyrethroid-resistant Aedes aegypti populations in Myanmar.

BACKGROUND: Single amino acid substitutions in the voltage-gated sodium channel associated with pyrethroid resistance constitute one of the main causative factors of knockdown resistance in insects. The kdr gene has been observed in several mosquito species; however, point mutations in the para gene of Aedes aegypti populations in Myanmar have not been fully characterized. The aim of the present study was to determine the types and frequencies of mutations in the para gene of Aedes aegypti collected from used tires in Yangon City, Myanmar. METHODOLOGY/PRINCIPAL FINDINGS: We determined high pyrethroid resistance in Aedes aegypti larvae at all collection sites in Yangon City, by using a simplified knockdown bioassay. We showed that V1016G and S989P mutations were widely distributed, with high frequencies (84.4% and 78.8%, respectively). By contrast, we were unable to detect I1011M (or I1011V) or L1014F mutations. F1534C mutations were also widely distributed, but with a lower frequency than the V1016G mutation (21.2%). High percentage of co-occurrence of the homozygous V1016G/S989P mutations was detected (65.7%). Additionally, co-occurrence of homozygous V1016G/F1534C mutations (2.9%) and homozygous V1016G/F1534C/S989P mutations (0.98%) were detected in the present study. CONCLUSIONS/SIGNIFICANCE: Pyrethroid insecticides were first used for malaria control in 1992, and have since been constantly used in Myanmar. This intensive use may explain the strong selection pressure toward Aedes aegypti, because this mosquito is generally a domestic and endophagic species with a preference for indoor breeding. Extensive use of DDT for malaria control before the use of this chemical was banned may also explain the development of pyrethroid resistance in Aedes aegypti.

Preventive effect of permethrin-impregnated long-lasting insecticidal nets on the blood feeding of three major pyrethroid-resistant malaria vectors in western Kenya.

BACKGROUND: Since the World Health Organization (WHO) adopted the use of long-lasting insecticidal nets (LLINs) as a principal strategy for effective malaria prevention and control, pyrethroids have been the only class of insecticides used for LLINs. The dramatic success of insecticide-treated nets (ITNs) and LLINs in African countries, however, has been threatened by the rapid development of pyrethroid resistance in vector mosquitoes. ITNs and LLINs are still used as effective self-protection measures, but there have been few studies on the effectiveness of ITNs and LLINs in areas where vector mosquitoes are pyrethroid-resistant. METHODS: To investigate the behavioral pattern of mosquitoes in the houses where LLINs were used, indoor mosquito trappings of Anopheles gambiae s.s., An. arabiensis, and An. funestus s.s. were performed with Centers for Disease Control and Prevention (CDC) miniature light trap equipped with a collection bottle rotator at 2-hour intervals between 4:00 pm and 8:00 am. The trapped female mosquitoes were identified and classified as unfed, blood fed, and gravid. The abdominal contents of fed female mosquitoes were used for DNA extractions to identify the blood source. RESULTS: A large proportion of human blood feeding of An. arabiensis and An. funestus s.s. (but not An. gambiae s.s.) took place during the time people were active outside LLINs. However, during the hours when people were beneath LLINs, these provided protective efficacy as indicated by reduced human blood feeding rates. CONCLUSION: LLINs provided effective protection against pyrethroid-resistant malaria vector populations during bedtime hours. However, protection of LLINs was insufficient during the hours when people were active outside of the bed nets. Such limitation of LLINs will need to be intensively addressed in African countries in the near future.