Anopheles Gambiae Sl Research Articles

Organophosphate and carbamate susceptibility profiling of Anopheles gambiae sl. across different ecosystems in southern Benin

Background To overcome the spread of high pyrethroid resistance in the main malaria vectors and malaria disease persistence, it is crucial to look for effective and better resistance management strategies. Understanding the phenotypic profile of Anopheles gambiae sl. against alternatives insecticides like organophosphates and carbamates is crucial. Methods Anopheles larvae and pupae were collected from the breeding sites in rice fields, pineapple crop areas, and peri-urban areas. WHO susceptibility tests were conducted on unfed female mosquitoes aged 3–5 days old. Mosquitoes were exposed to malathion 5%, pirimiphos-methyl 0.25%, and bendiocarb 0.1% using the standard WHO protocol. Polymerase chain reaction (PCR) techniques were used to detect species, kdr and Ace-1 mutations. Results Anopheles gambiae sl. from Sèdjè-Dénou rice field population was resistant to bendiocarb (0.1%) with a mortality rate of 72.2% whereas Anopheles gambiae sl. populations from Zinvié-Dokomey (rice field), Zè-Tozounmè (pineapple field), and Adjagbo (peri-urban area) were suspected to be resistant with mortality rates of 90%, 93.5%, 95.4% respectively. However, all of them were susceptible to organophosphates (malathion and pirimiphos-methyl) with a mortality rate of 100%. PCR assay revealed that 100% of the mosquitoes tested were Anopheles coluzzii. The frequencies of Ace-1R mutation in all Anopheles coluzzii populations tested were low (3–27%). Conclusions Organophosphates (malathion and pirimiphos-methyl) have maintained their efficacy against Anopheles coluzzii populations from Sèdjè-Dénou (rice field), Zè Tozounmè (pineapple field), Zinvié Dokomey (rice field), or Adjagbo (peri-urban area). The good efficacy of these organophosphates against Anopheles coluzzii populations from the southern part of Benin are observed in the current study. The use of pirimiphos-methyl for IRS in this part of the country would be a successful alternative for malaria control in this area.

Community evaluation of the physical and insecticidal durability of DuraNet® Plus, an alpha-cypermethrin and piperonyl butoxide incorporated mosquito net: protocol for a multi-country study in West, Central and East Africa

BackgroundPyrethroid-PBO nets have demonstrated improved impact against clinical malaria transmitted by pyrethroid resistant mosquito vectors and are being scaled up across Africa. However very little is known about their physical and insecticidal durability under operational conditions. This study will investigate the attrition, fabric integrity, insecticide content and bioefficacy of DuraNet® Plus, a new WHO prequalified alphacypermethrin and PBO incorporated net developed by Shobikaa Impex Private Limited over 3 years of field use in communities in Benin, Cameroon and Tanzania.MethodsThe study will be conducted in parallel in selected villages in Zakpota District in Benin, Mbalmayo, District in Cameroon and Muheza District in Tanzania. In each country, ~ 1800 households will be recruited and randomised to receive DuraNet® Plus or DuraNet® (a WHO prequalified alphacypermethrin-only ITN). Follow up surveys will be performed at 1 month post distribution to investigate adverse events and subsequently every 6–12 months to assess ITN attrition and fabric integrity following standard WHO procedures. A second cohort of nets will be withdrawn every 6–12 months and assessed for alpha-cypermethrin and PBO content and for entomological activity in laboratory bioassays (cone bioassays and tunnel tests). Alpha-cypermethrin bioefficacy will be monitored using the susceptible Anopheles gambiae Kisumu strain in cone bioassays while PBO bioefficacy will be monitored using pyrethroid resistant strains with overexpressed P450 enzymes in tunnel tests to determine the proportion of efficacious nets (≥ 95% knockdown, ≥ 80% mortality or ≥ 90% blood feeding inhibition in tunnels) at each time point. Nets withdrawn at 12, 24 and 36 months from each country will also be tested in experimental hut trials against wild free-flying pyrethroid resistant Anopheles gambiae sl in Côvè Benin to investigate the superiority of DuraNet® Plus over DuraNet® at each time point under semi field conditions.ConclusionThis large-scale multi country trial will provide useful information on the durability of a pyrethroid-PBO net (DuraNet® Plus) in 3 different regions in sub-Saharan Africa. The methods proposed for bioefficacy testing could also contribute towards the development of new standardised guidelines for monitoring the insecticidal efficacy of pyrethroid-PBO nets under operational conditions.

Resistant status and intensity of resistance of Anopheles gambiae sensu lato to pyrethroid and organophosphate in Lafia, Nasarawa State Nigeria

Monitoring and understanding the trend and dynamics of insecticide resistance are key to devising efficient control strategies. This study was carried out to investigate the resistance status of Anopheles gambiae sl mosquitoes in Lafia, Nasarawa State Nigeria to three pyrethroid (deltamethrin, Alphacypermethrin and permethrin) with concentration of 12.5 µg/bottle each and an organophosphate (pirimiphose-methyl 20 µg/bottle). Larvae and pupae of Anopheles mosquito were sampled within the metropolis and reared to adults in the insectary of Nasarawa State University, Keffi Nasarawa State. Emerged adults of 2 to 5 days old and non-blood fed were tested for resistance using CDC bottle bioassay susceptibility protocol. Results showed that the local mosquitoes species were resistant to all the three pyrethroid (deltamethrin, Alphacypermethrin and permethrin) but showed possible resistant to organophosphate (pirimiphose-methyl) with mortalities of 88, 84, 54, and 95% respectively. Exposure to piperonyl butoxide (PBO) as a synergist resulted in the susceptibility (100 and 98%) of mosquitoes to deltamethrin and Alphacypermethrin respectively but resistant (81%) to permethrin. The intensity of resistance of the vector population to pyrethroid was high especially with permethrin. The mosquitoes were only susceptible (100%) to deltamethrin and Alphacypermethrin at X5 concentration and permethrin at X10 concentration. Insecticide based intervention strategy remain the principal vector control measure in malaria endemic countries like Nigeria. Based on the result of this study, future vector control programme should take into consideration the deployment of nets that are treated with alphacypermethrin or deltamethrin combined with PBO (PBO based nets) in the study area or increase the concentration of the pyrethoid insecticides used in this study on long lasting nets after their safety to human is assured.

Larvicidal activity in the laboratory of aqueous extracts of lantana camara, Bougainvillea spectabilisand Tephrosia villosa against Anopheles gambiae SL vector of malaria in Cote d’Ivoire

Considering the pollution and resistance of chemical insecticides in Anopheles gambiae S.L., alternative control approaches have led to this study. Larvicidal activities of the leaves of Lantana camara, Bougainvillea spectabilis and Tephrosia villosa against the fourth instar larvae of Anopheles gambiae S.L. were evaluated. The susceptibility level and the homogeneity of wild strains were determined according to the method of Sinègre. The highest mortality rates were obtained from 15mg/ml with Lantana camara and Bougainvillea spectabilis. The larvicidal activity of Lantana camara on the wild strains was higher than Bougainvillea spectabilis. These wild strains have the same sensitivity as well as the KISUMU strain towards Bougainvillea spectabilis and Lantana camara. However, the wild population response to aqueous extracts was homogeneous against Bougainvillea spectabilis and heterogeneous against Lantana camara. Lantana camara and Bougainvillea spectabilis leaves were the most active aqueous extract on larvae of Anopheles gambiae S.L.

Transcriptomic analysis of resistance and short-term induction response to pyrethroids, in Anopheles coluzzii legs

BackgroundInsecticide-treated bed nets and indoor residual spraying comprise the major control measures against Anopheles gambiae sl, the dominant vector in sub-Saharan Africa. The primary site of contact with insecticide is through the mosquitoes’ legs, which represents the first barrier insecticides have to bypass to reach their neuronal targets. Proteomic changes and leg cuticle modifications have been associated with insecticide resistance that may reduce the rate of penetration of insecticides. Here, we performed a multiple transcriptomic analyses focusing on An. coluzzii legs.ResultsFirstly, leg-specific enrichment analysis identified 359 genes including the pyrethroid-binder SAP2 and 2 other chemosensory proteins, along with 4 ABCG transporters previously shown to be leg enriched. Enrichment of gene families included those involved in detecting chemical stimuli, including gustatory and ionotropic receptors and genes implicated in hydrocarbon-synthesis.Subsequently, we compared transcript expression in the legs of a highly resistant strain (VK7-HR) to both a strain with very similar genetic background which has reverted to susceptibility after several generations without insecticide pressure (VK7-LR) and a lab susceptible population (NG). Two hundred thirty-two differentially expressed genes (73 up-regulated and 159 down-regulated) were identified in the resistant strain when compared to the two susceptible counterparts, indicating an over-expression of phase I detoxification enzymes and cuticular proteins, with decrease in hormone-related metabolic processes in legs from the insecticide resistant population.Finally, we analysed the short-term effect of pyrethroid exposure on An. coluzzii legs, comparing legs of 1 h-deltamethrin-exposed An. coluzzii (VK7-IN) to those of unexposed mosquitoes (VK7-HR) and identified 348 up-regulated genes including those encoding for GPCRs, ABC transporters, odorant-binding proteins and members of the divergent salivary gland protein family.ConclusionsThe data on An. coluzzii leg-specific transcriptome provides valuable insights into the first line of defense in pyrethroid resistant and short-term deltamethrin-exposed mosquitoes. Our results suggest that xenobiotic detoxification is likely occurring in legs, while the enrichment of sensory proteins, ABCG transporters and cuticular genes is also evident. Constitutive resistance is primarily associated with elevated levels of detoxification and cuticular genes, while short-term insecticide-induced tolerance is linked with overexpression of transporters, GPCRs and GPCR-related genes, sensory/binding and salivary gland proteins.

Entomological characteristics of mosquitoes breeding sites in two areas of the town of Douala, Cameroon

Many infectious diseases including malaria, lymphatic filariasis, chikungunya and dengue are transmitted by mosquitoes. Understanding the entomological diversity in a given area is critical for defining health risks, but also to design and evaluate control measures. Our study aimed to establish an entomological database of the Culicidae fauna in two clusters (Nyalla and Kambo) in the city of Douala in Cameroon. A longitudinal study was conducted from August to December in the Nyalla and Kambo neighbourhoods in the city of Douala. Culicidae larvae were weekly collected and reared until adult emergence. Adults were then morphologically identified using reference identification keys. A total of 20 breeding sites were productive out of an approximately 90 sites prospected. Water collections, pits, swamps and cinder blocks were main categories of larval habitats. In total, 2125 mosquito larvae were collected and reared during the survey. A total of 1790 mosquito adults emerged and were further identified and distributed among three genera: Culex (73.3%); Anopheles (22.6%) and Aedes (4.1%). Eleven Culicidae species were identified: Culex duttoni, Cx. descens, Cx. poicilipes, Cx. univittatus, Cx tigripes, Cx. antennatus, Cx. simpsoni, Cx. pipiens, Anopheles gambiae sl, Aedes aegypti and Ae. albopictus. Culex duttoni (39%) and An. gambiae s.l. (22.6%) were the main species recorded. The equitability, species richness and abundance of these genera was influenced by the nature of breeding sites and physical parameters including rainfall and temperature. The present study provides insights on the entomological diversity of the Culicidae fauna in the study area. Such information will help to orientate the fight against mosquito vectors in conjunction with the species and the types of breeding sites.

ENTOMOLOGICAL SURVEY OF MOSQUITO VECTORS OF LYMPHATIC FILARIASIS IN TALATAN-MAFARA AND TSAFE LOCAL GOVERNMENT AREAS OF ZAMFARA STATE, NIGERIA

Entomological survey of mosquito vectors was carried out to determine species abundance and identify those responsible for the transmission of lymphatic filariasis in Talatan-mafara and Tsafe Local Government Areas of Zamfara State, Nigeria. Houses were randomly selected for mosquito collection. Aerosol (Baygon) was sprayed in the rooms to knockdown indoor resting mosquitoes. A total of 5,230 mosquitoes comprising of 3,104 females and 2,126 males were collected and 1,182 engorged females were dissected to isolate filarial worm, Wuchereria bancrofti. The species of mosquitoes encountered include Culex quinquefasciatus (84%), Anopheles funestus (10%) and Anopheles gambiae sl. (6%). The overall infection and infectivity rates of the mosquitoes with Wuchereria bancrofti were 1.86% and 1.44% respectively. The infection/infectivity rates of Culex quinquefasciatus, Anopheles funestus and Anopheles gambiae sl. were 1.01%/0.51%, 2.97%/2.97% and 4.54%/4.14% respectively.

Evaluation of Actin-1 Expression in Wild Caught Wuchereria bancrofti-Infected Mosquito Vectors.

Background Wuchereria bancrofti is the major cause of lymphatic filariasis transmitted by mosquito vectors. In the vector-parasite interaction and among other proteins, actin-1 has been implicated for successful transmission of the pathogen in laboratory-controlled experiments. However, validation of this finding from the pathogen's natural environment is required. Objective This study is aimed at evaluating actin-1 expression upon Wuchereria bancrofti infection in mosquito vectors collected during an epidemiology study in Tsafe Local Government Area of Zamfara State, Nigeria. Methods Mosquitoes were collected and identified using morphological keys, which include length of maxillary palps, pale spots on the wings, and scale patterns on the abdomen. This was followed by detection of the 188 bp SspI marker of Wuchereria bancrofti infection using polymerase chain reaction (PCR). The mRNA levels of the actin-1 gene were evaluated in the infected Anopheles gambiae sl and Culex quinquefasciatus and their controls, which were adult reared from the larvae in the study area. Results The mosquitoes were identified to be Anopheles gambiae sl and Culex quinquefasciatus, while infection by Wuchereria bancrofti was confirmed by amplification of the 188 bp SspI marker. A 4.85 and 4.09 relative fold increase in actin-1 gene expression in Wuchereria bancrofti-infected Anopheles gambiae sl and Culex quinquefasciatus was observed. Thus, for the first time we reported that the actin-1 gene in wild caught mosquito vectors (Anopheles gambiae sl and Culex quinquefasciatus) infected with Wuchereria bancrofti is upregulated. Conclusion The actin-1 gene is upregulated and similarly expressed during W. bancrofti infection in mosquito vectors in the study area and this may likely serve as a biomarker and viable strategy for the control of parasite transmission in endemic areas.

Impact of Insecticide Resistance on P. falciparum Vectors' Biting, Feeding, and Resting Behaviour in Selected Clusters in Teso North and South Subcounties in Busia County, Western Kenya.

Introduction Behavioural resistance to insecticides restrains the efficacy of vector control tools against mosquito-transmitted diseases. The current study is aimed at determining the impact of insecticide resistance on major malaria vectors' biting, feeding, and resting behaviour in areas with and areas without insecticide resistance in Teso North and Teso South, Busia County, Western Kenya. Methods Mosquito larvae were sampled using a dipper, reared into 3-5-day-old female mosquitoes [4944] which were exposed to 0.75% permethrin and 0.05% deltamethrin using World Health Organization tube assay method. Blood meal, species identification, and kdr Eastgene PCRs were also performed on adult mosquitoes sampled using mosquito collection methods [3448]. Biting, feeding, resting, and exiting behaviours of field-collected mosquitoes from five selected clusters were analysed. Results The lowest Kdr genotypic frequency (SS) proportion was found in female Anophelines collected in Kengatunyi at 58% while Rwatama had the highest genotypic frequency at 93%, thus susceptible and resistant clusters, respectively. The peak hour for mosquito seeking a human bite was between 0300 and 0400 hrs in the resistant cluster and 0400-0500 hrs in the susceptible cluster. The heterozygous mosquitoes maintained the known 2100-2200 hrs peak hour. There was a higher proportion of homozygous susceptible vectors (86.4%) seeking humans indoor than outdoor bitters (78.3%). Mosquito blood meals of human origin were 60% and 87% in susceptible Kengatunyi and resistant Rwatama cluster, respectively. There was significant difference between homozygous-resistant vectors feeding on human blood compared to homozygous susceptible mosquitoes (p ≤ 0.05). The proportion of bovine blood was highest in the susceptible cluster. A higher proportion of homozygous-resistant anophelines were feeding and resting indoors. No heterozygous mosquito was found resting indoor while 4.2% of the mosquitoes were caught while exiting the house through the window. Discussion. A shift in resistant Anopheles gambiae sl highest peak hour of aggressiveness from 2100-2200 hrs to 0300-0400 hrs is a key change in its biting pattern. Due to the development of resistance, mosquitoes no longer have to compete against the time the human host enters into the formerly lethal chemical and or physical barrier in the form of long-lasting insecticide-treated net. No heterozygous LS mosquito rested indoors possibly due to disadvantages of heterozygosity which could have increased their fitness costs as well as energy costs in the presence of the insecticidal agents in the treated nets. Conclusions and recommendations. Out of bed biting by female mosquitoes and partial susceptibility may contribute to residual malaria transmission. Insecticide-resistant vectors have become more endophagic and anthropophillic. Hence, insecticidal nets, zooprophylaxis, and novel repellents are still useful chemical, biological, and physical barriers against human blood questing female mosquitoes. Further studies should be done on genetic changes in mosquitoes and their effects on changing mosquito behaviour.

English

Bancroftian filariasis in Nigeria is endemic with 22.1% of the population thought to be infected. The main mosquito genera implicated with Wuchereria bancrofti transmission are Anopheles and Culex. The study was carried out to compare the infectivity rates of the vectors between the high transmission (rainy) and the low transmission (dry) seasons. Mosquitoes were sampled from houses and compared from six sentinel villages (3 each from Ohaukwu and Abakiliki Local Government Areas) of Ebonyi State, Nigeria. Day resting indoor collection (DRI) by Aspirator and Pyrethrum Spray Catch (PSC) were used to collect mosquitoes between 7:00 and 11:00am. After morphological identification, female parous mosquitoes were dissected in search for infective (L3) larvae of W. bancrofti. A total of 4,840 female mosquitoes were dissected. More mosquitoes were caught in the rainy season than in the dry season. Infectivity rates of vectors in Ohaukwu villages were 3.54 and 5.41% in the rainy and dry seasons, respectively, whereas in Abakiliki villages these were 1.85 and 1.19%, respectively. There was no significant difference in the overall infectivity rates between the two seasons in both Ohaukwu and Abakiliki villages (p>0.05). Similarly, no significant difference in the total/average transmission potentials were found between the seasons (p>0.05). Anopheles gambiae sl was the main vector in both study sites followed by an Anopheles funestus and Culex quinquefasciatus. There was a difference in infectivity rates of Anopheles species between the wet and dry seasons (p<0.05), whereas no significant difference exist in infectivity rates of Anopheles species and Cx. quinquefasciatus (p>0.05). Findings were discussed in the context of on-going plans to eliminate filariasis and the transmitting vectors.   Key words: Bancroftian filariasis, rainy season, dry season, Wuchereria bancrofti.&nbsp

Suscepibility of Anopheles gambiae sl and Culex quinquefasciatus to diverse insecticides in the city of N'Djamena.

Mosquito resistance to insecticides remains a major concern for vector control programs based on chemical methods. Hence, a thorough knowledge of vector susceptibility to products recommended for public health is required for effective vector control programs. In this study, we assessed the susceptibility of wild Anopheles gambiae sl and Culex quinquefasciatus populations from N'Djamena (Chad) to four insecticide classes recommended for public health. The study took place from July to October 2014. Following WHO guidelines, bioassays were performed on two- to four-day-old, non-engorged female mosquitoes of both species, collected in the field. The insecticides used were deltamethrin (0.05%), permethrin (1%), DDT (4%), bendiocarb (0.01%), and malathion (5%). Anopheles gambiae sl showed full susceptibility to bendiocarb and malathion but was resistant to pyrethroids; mortality rates were 2% for deltamethrin, 2% for permethrin, and 0% for DDT. Culex quinquefasciatus was susceptible to malathion but resistant to the other insecticides, with a mortality rate of 22% for deltamethrin, 29% for permethrin, 23% for DDT, and 45% bendiocarb. The resistance of Anopheles gambiae sl and of Culex quinquefasciatus may affect the population's adherence to the use of insecticide-treated mosquito nets. Malaria control programs are advised to extend insecticide resistance monitoring to the main culicide species that bite human populations.

Indoor residual spraying with a mixture of clothianidin (a neonicotinoid insecticide) and deltamethrin provides improved control and long residual activity against pyrethroid resistant Anopheles gambiae sl in Southern Benin.

IntroductionThere is an urgent need for new insecticides for indoor residual spraying (IRS) which can provide improved and prolonged control of malaria vectors that have developed resistance to existing insecticides. The neonicotinoid, clothianidin represents a class of chemistry new to public health. Clothianidin acts as an agonist on nicotinic acetyl choline receptors. IRS with a mixture of Clothianidin and another WHO approved insecticide such as deltamethrin could provide improved control of insecticide resistant malaria vector populations and serve as a tool for insecticide resistance management.MethodsThe efficacy and residual activity of a novel IRS mixture of deltamethrin and clothianidin was evaluated against wild pyrethroid resistant An. gambiae sl in experimental huts in Cove, Benin. Two application rates of the mixture were tested and comparison was made with clothianidin and deltamethrin applied alone. To assess the residual efficacy of the treatments on different local wall substrates, the inner walls of the experimental huts were covered with either cement, mud or plywood.ResultsClothianidin demonstrated a clear delayed expression in mortality of wild pyrethroid resistant An. gambiae sl in the experimental huts which reached its full effect 120 hours after exposure. Overall mortality over the 12-month hut trial was 15% in the control hut and 24–29% in the deltamethrin-treated huts. The mixture of clothianidin 200mg/m2 and deltamethrin 25mg/m2 induced high overall hut mortality rates (87% on mud walls, 82% on cement walls and 61% on wooden walls) largely due to the clothianidin component and high hut exiting rates (67–76%) mostly due to the deltamethrin component. Mortality rates remained >80% for 8–9 months on mud and cement walls. The residual activity trend was confirmed by results from monthly in situ cone bioassays with laboratory susceptible An. gambiae Kisumu strain.ConclusionIRS campaigns with the mixture of clothianidin plus deltamethrin have the potential to provide prolonged control of malaria transmitted by pyrethroid resistant mosquito populations.

Chlorfenapyr (A Pyrrole Insecticide) Applied Alone or as a Mixture with Alpha-Cypermethrin for Indoor Residual Spraying against Pyrethroid Resistant Anopheles gambiae sl: An Experimental Hut Study in Cove, Benin

BackgroundIndoor spraying of walls and ceilings with residual insecticide remains a primary method of malaria control. Insecticide resistance in malaria vectors is a growing problem. Novel insecticides for indoor residual spraying (IRS) which can improve the control of pyrethroid resistant malaria vectors are urgently needed. Insecticide mixtures have the potential to improve efficacy or even to manage resistance in some situations but this possibility remains underexplored experimentally. Chlorfenapyr is a novel pyrrole insecticide which has shown potential to improve the control of mosquitoes which are resistant to current WHO-approved insecticides.MethodThe efficacy of IRS with chlorfenapyr applied alone or as a mixture with alpha-cypermeththrin (a pyrethroid) was evaluated in experimental huts in Cove, Southern Benin against wild free flying pyrethroid resistant Anopheles gambiae sl. Comparison was made with IRS with alpha-cypermethrin alone. Fortnightly 30-minute in situ cone bioassays were performed to assess the residual efficacy of the insecticides on the treated hut walls.ResultsSurvival rates of wild An gambiae from the Cove hut site in WHO resistance bioassays performed during the trial were >90% with permethrin and deltamethrin treated papers. Mortality of free-flying mosquitoes entering the experimental huts was 4% in the control hut. Mortality with alpha-cypermethrin IRS did not differ from the control (5%, P>0.656). The highest mortality was achieved with chlorfenapyr alone (63%). The alpha-cypermethrin + chlorfenapyr mixture killed fewer mosquitoes than chlorfenapyr alone (43% vs. 63%, P<0.001). While the cone bioassays showed a more rapid decline in residual mortality with chlorfenapyr IRS to <30% after only 2 weeks, fortnightly mortality rates of wild free-flying An gambiae entering the chlorfenapyr IRS huts were consistently high (50–70%) and prolonged, lasting over 4 months.ConclusionIRS with chlorfenapyr shows potential to significantly improve the control of malaria transmission in pyrethroid resistant areas compared to pyrethroid IRS or the mixture. Thirty minute in situ cone bioassays are not predictive of the performance of chlorfenapyr IRS under field conditions.

Evaluation of Field Performance of Insecticide-Treated Mosquito Nets in North-Western Burkina Faso

Aims: The aim of the study was to evaluate the field performance of insecticide-treated mosquito nets (ITNs) in north-western Burkina Faso. Study design: Cross-sectional survey. Place and Duration: The study took place between September and November 2008 (end of rainy season) in rural north-western Burkina Faso. Methodology: Standard WHO bioassays were performed on field-collected ITNs from three areas of different insecticide pressure: semi-urban Nouna town, three villages with cotton agriculture, and three villages without cotton agriculture. Data on age and washing Short Communication International Journal of TROPICAL DISEASE & Health, 2(4): 284-289, 2012 285 frequency of ITNs was collected, and deltamethrin content was determind by HPLC. Results: The mean age of field-collected ITNs was 2.1 years. The mosquito mortality rate (Anopheles gambiae sl.) after 24 hours was 4% for the negative controls, 90% for the positive controls, and 73% for field-used ITNs. Differences in mosquito mortality between sub-areas disappeared after controlling for confounding variables. Conclusion: This study demonstrates that under real life conditions, deltamethrin still shows some level of effectiveness despite ongoing insecticide pressure. However, deltamethrin resistance has been observed in other parts of the country and thus close surveillance of ITN efficacy is needed. More frequent replacement of ITNs is also recommended.

A Simple Chelex Protocol for DNA Extraction from &lt;em&gt;Anopheles&lt;/em&gt; spp.

Endemic countries are increasingly adopting molecular tools for efficient typing, identification and surveillance against malaria parasites and vector mosquitoes, as an integral part of their control programs1,2,3,4,5. For sustainable establishment of these accurate approaches in operations research to strengthen malaria control and elimination efforts, simple and affordable methods, with parsimonious reagent and equipment requirements are essential6,7,8. Here we present a simple Chelex-based technique for extracting malaria parasite and vector DNA from field collected mosquito specimens. We morphologically identified 72 Anopheles gambiae sl. from 156 mosquitoes captured by pyrethrum spray catches in sleeping rooms of households within a 2,000 km2 vicinity of the Malaria Institute at Macha. After dissection to separate the head and thorax from the abdomen for all 72 Anopheles gambiae sl. mosquitoes, the two sections were individually placed in 1.5 ml microcentrifuge tubes and submerged in 20 μl of deionized water. Using a sterile pipette tip, each mosquito section was separately homogenized to a uniform suspension in the deionized water. Of the ensuing homogenate from each mosquito section, 10 μl was retained while the other 10 μl was transferred to a separate autoclaved 1.5 ml tube. The separate aliquots were subjected to DNA extraction by either the simplified Chelex or the standard salting out extraction protocol9,10. The salting out protocol is so-called and widely used because it employs high salt concentrations in lieu of hazardous organic solvents (such as phenol and chloroform) for the protein precipitation step during DNA extraction9. Extracts were used as templates for PCR amplification using primers targeting arthropod mitochondrial nicotinamide adenine dinucleotide dehydrogenase (NADH) subunit 4 gene (ND4) to check DNA quality11, a PCR for identification of Anopheles gambiae sibling species10 and a nested PCR for typing of Plasmodium falciparum infection12. Comparison using DNA quality (ND4) PCR showed 93% sensitivity and 82% specificity for the Chelex approach relative to the established salting out protocol. Corresponding values of sensitivity and specificity were 100% and 78%, respectively, using sibling species identification PCR and 92% and 80%, respectively for P. falciparum detection PCR. There were no significant differences in proportion of samples giving amplicon signal with the Chelex or the regular salting out protocol across all three PCR applications. The Chelex approach required three simple reagents and 37 min to complete, while the salting out protocol entailed 10 different reagents and 2 hr and 47 min' processing time, including an overnight step. Our results show that the Chelex method is comparable to the existing salting out extraction and can be substituted as a simple and sustainable approach in resource-limited settings where a constant reagent supply chain is often difficult to maintain.

A Simple Chelex Protocol for DNA Extraction from &lt;em&gt;Anopheles&lt;/em&gt; spp.

Endemic countries are increasingly adopting molecular tools for efficient typing, identification and surveillance against malaria parasites and vector mosquitoes, as an integral part of their control programs1,2,3,4,5. For sustainable establishment of these accurate approaches in operations research to strengthen malaria control and elimination efforts, simple and affordable methods, with parsimonious reagent and equipment requirements are essential6,7,8. Here we present a simple Chelex-based technique for extracting malaria parasite and vector DNA from field collected mosquito specimens.We morphologically identified 72 Anopheles gambiae sl. from 156 mosquitoes captured by pyrethrum spray catches in sleeping rooms of households within a 2,000 km2 vicinity of the Malaria Institute at Macha. After dissection to separate the head and thorax from the abdomen for all 72 Anopheles gambiae sl. mosquitoes, the two sections were individually placed in 1.5 ml microcentrifuge tubes and submerged in 20 μl of deionized water. Using a sterile pipette tip, each mosquito section was separately homogenized to a uniform suspension in the deionized water. Of the ensuing homogenate from each mosquito section, 10 μl was retained while the other 10 μl was transferred to a separate autoclaved 1.5 ml tube. The separate aliquots were subjected to DNA extraction by either the simplified Chelex or the standard salting out extraction protocol9,10. The salting out protocol is so-called and widely used because it employs high salt concentrations in lieu of hazardous organic solvents (such as phenol and chloroform) for the protein precipitation step during DNA extraction9.Extracts were used as templates for PCR amplification using primers targeting arthropod mitochondrial nicotinamide adenine dinucleotide dehydrogenase (NADH) subunit 4 gene (ND4) to check DNA quality11, a PCR for identification of Anopheles gambiae sibling species10 and a nested PCR for typing of Plasmodium falciparum infection12. Comparison using DNA quality (ND4) PCR showed 93% sensitivity and 82% specificity for the Chelex approach relative to the established salting out protocol. Corresponding values of sensitivity and specificity were 100% and 78%, respectively, using sibling species identification PCR and 92% and 80%, respectively for P. falciparum detection PCR. There were no significant differences in proportion of samples giving amplicon signal with the Chelex or the regular salting out protocol across all three PCR applications. The Chelex approach required three simple reagents and 37 min to complete, while the salting out protocol entailed 10 different reagents and 2 hr and 47 min' processing time, including an overnight step. Our results show that the Chelex method is comparable to the existing salting out extraction and can be substituted as a simple and sustainable approach in resource-limited settings where a constant reagent supply chain is often difficult to maintain.

The effect of repeated washing of long-lasting insecticide-treated nets (LLINs) on the feeding success and survival rates of Anopheles gambiae

BackgroundInsecticide-treated nets protect users from mosquito bites, thereby preventing transmissions of mosquito borne pathogens. Repeated washing of nets removes insecticide on the netting rendering them ineffective within a short period. Long-lasting insecticide-treated nets (LLINs) offer longer time protection against such bites because they are more wash resistant, and are preferred to conventionally treated nets. However, there is limited information on the effect of repeated washing of LLINs on the feeding success and survival of wild malaria vectors.MethodsThe current study evaluated the effect of repeated washing of four brands of LLINs on the feeding success and survival rates of Anopheles gambiae sl reared from wild strains. In this study, two- to five-day old F1s, reared from gravid mosquitoes collected from an area with a high coverage of LLINs were offered blood meals through protective barriers of the above LLINs. Mosquitoes were exposed for a period of 10 minutes each time. Nets were tested unwashed and subsequently after every 5th through wash 15. After exposure mosquitoes were sorted out according to their feeding status. They were then held under normal laboratory conditions for 24 hours and mortality was scored in both fed and unfed.ResultsIt was observed that mosquitoes did not feed through a barrier of unwashed LLINs. However, the feeding success and survival rates increased with successive number of washes and were also net brand dependant. After 15 washes, 49% of vectors succeeded to feed through a protective barrier of PermaNet 2.0 and 50% of the fed died after 24 hrs while after the same number of washes 60% of vectors succeeded to feed through Olyset brand of LLINs and all of them survived. In general, more mosquitoes survived after feeding through Olyset compared to the other four brands that were evaluated. When efficacy of individual LLINs was compared by a t-test analysis to a conventionally treated net, the results were not significantly different statistically for Olyset (p = 0.239) and NetProtect (TNT) (p = 0.135). However, the results were highly significant when comparison was made with PermaNet and Interceptor (BASF); p values 0.015 and 0.025 respectively.ConclusionThe result of this study shows that repeated washing of LLINs at short time intervals using local washing methods may render them infective within a short time in preventing local vectors from feeding.

Polymorphismes érythrocytaires au Mali : épidémiologie et mécanismes de résistance contre la progression vers les formes graves du paludisme à Plasmodium falciparum

Homo sapiens and Plasmodium falciparum have co-evolved since the beginning of agriculture, 10,000 to 20,000 years ago. By domesticating plants and animals, humans linked their destiny to one of the main vectors of malaria, Anopheles gambiae sl complex. The biological interaction between these three species led to exchanges of genes and biochemical processes with significant mutual influence. Humans acquired mutations with selective protective advantages against serious and fatal forms of this hemosporidiosis. This is the case of hemoglobin S, hemoglobin C, hemoglobin E, thalassemias, ovalocytosis and G6PD deficiency, among others. Many epidemiological studies published since 1949 have shown a geographic link between malaria and certain erythrocyte polymorphisms. The link with hemoglobin C was discovered only recently, in 2000, initially in Mali in the Dogon population, then in Burkina Faso. Epidemiological and molecular and cellular biology studies done in Mali and elsewhere showed that the C and S alleles, and G6PD deficiency [A-], conferred significant protection against lethal forms of Plasmodium falciparum malaria. Molecular genetic studies, based on functional genomics, transcriptomics and proteomics, provided possible explanations. Advances in molecular biology and a better understanding of the immune mechanisms underlying this protection will hopefully lead to the development of effective second- and third-generation malaria vaccines. Epidemiological and fundamental research efforts have identified some of the mechanisms by which these erythrocyte polymorphisms protect against the most lethal hematozoan parasite, Plasmodium falciparum.