High Level of Pyrethroid Resistance in an Anopheles funestus Population of the Chokwe District in Mozambique

Background

Although Anopheles funestus is difficult to rear, it is crucial to analyse field populations of this malaria vector in order to successfully characterise mechanisms of insecticide resistance observed in this species in Africa. In this study we carried out a large-scale field collection and rearing of An. funestus from Mozambique in order to analyse its susceptibility status to insecticides and to broadly characterise the main resistance mechanisms involved in natural populations.

Methodology/Principal Findings

3,000 F₁ adults were obtained through larval rearing. WHO susceptibility assays indicated a very high resistance to pyrethroids with no mortality recorded after 1h30min exposure and less than 50% mortality at 3h30min. Resistance to the carbamate, bendiocarb was also noted, with 70% mortality after 1h exposure. In contrast, no DDT resistance was observed, indicating that no kdr-type resistance was involved. The sequencing of the acetylcholinesterase gene indicated the absence of the G119S and F455W mutations associated with carbamate and organophosphate resistance. This could explain the absence of malathion resistance in this population. Both biochemical assays and quantitative PCR implicated up-regulated P450 genes in pyrethroid resistance, with GSTs playing a secondary role. The carbamate resistance observed in this population is probably conferred by the observed altered AChE with esterases also involved.

Conclusion/Significance

The high level of pyrethroid resistance in this population despite the cessation of pyrethroid use for IRS in 1999 is a serious concern for resistance management strategies such as rotational use of insecticides. As DDT has now been re-introduced for IRS, susceptibility to DDT needs to be closely monitored to prevent the appearance and spread of resistance to this insecticide.     

Exploring mechanisms of multiple insecticide resistance in a population of the malaria vector Anopheles funestus in Benin

Background

The insecticide resistance status of the malaria vector Anopheles funestus and the underlying resistance mechanisms remain uncharacterised in many parts of Africa, notably in Benin, West Africa. To fill this gap in our knowledge, we assessed the susceptibility status of a population of this species in Pahou, Southern Benin and investigated the potential resistance mechanisms.

Methodology/Principal Findings

WHO bioassays revealed a multiple resistance profile for An. funestus in Pahou. This population is highly resistant to DDT with no mortality in females after 1h exposure to 4%DDT. Resistance was observed against the Type I pyrethroid permethrin and the carbamate bendiocarb. A moderate resistance was detected against deltamethrin (type II pyrethroids). A total susceptibility was observed against malathion, an organophosphate. Pre-exposure to PBO did not change the mortality rates for DDT indicating that cytochrome P450s play no role in DDT resistance in Pahou. No L1014F kdr mutation was detected but a correlation between haplotypes of two fragments of the Voltage-Gated Sodium Channel gene and resistance was observed suggesting that mutations in other exons may confer the knockdown resistance in this species. Biochemical assays revealed elevated levels of GSTs and cytochrome mono-oxygenases in Pahou. No G119S mutation and no altered acetylcholinesterase gene were detected in the Pahou population. qPCR analysis of five detoxification genes revealed that the GSTe2 is associated to the DDT resistance in this population with a significantly higher expression in DDT resistant samples. A significant over-expression of CYP6P9a and CYP6P9b previously associated with pyrethroid resistance was also seen but at a lower fold change than in southern Africa.

Conclusion

The multiple insecticide resistance profile of this An. funestus population in Benin shows that more attention should be paid to this important malaria vector for the implementation and management of current and future malaria vector control programs in this country.     

Pyrethroid Resistance in an Anopheles funestus Population from Uganda

Background

The susceptibility status of Anopheles funestus to insecticides remains largely unknown in most parts of Africa because of the difficulty in rearing field-caught mosquitoes of this malaria vector. Here we report the susceptibility status of the An. funestus population from Tororo district in Uganda and a preliminary characterisation of the putative resistance mechanisms involved.

Methodology/Principal Findings

A new forced egg laying technique used in this study significantly increased the numbers of field-caught females laying eggs and generated more than 4000 F₁ adults. WHO bioassays indicated that An. funestus in Tororo is resistant to pyrethroids (62% mortality after 1 h exposure to 0.75% permethrin and 28% mortality to 0.05% deltamethrin). Suspected DDT resistance was also observed with 82% mortality. However this population is fully susceptible to bendiocarb (carbamate), malathion (organophosphate) and dieldrin with 100% mortality observed after exposure to each of these insecticides. Sequencing of a fragment of the sodium channel gene containing the 1014 codon conferring pyrethroid/DDT resistance in An. gambiae did not detect the L1014F kdr mutation but a correlation between haplotypes and resistance phenotype was observed indicating that mutations in other exons may be conferring the knockdown resistance in this species. Biochemical assays suggest that resistance in this population is mediated by metabolic resistance with elevated level of GSTs, P450s and pNPA compared to a susceptible strain of Anopheles gambiae. RT-PCR further confirmed the involvement of P450s with a 12-fold over-expression of CYP6P9b in the Tororo population compared to the fully susceptible laboratory colony FANG.

Conclusion

This study represents the first report of pyrethroid/DDT resistance in An. funestus from East Africa. With resistance already reported in southern and West Africa, this indicates that resistance in An. funestus may be more widespread than previously assumed and therefore this should be taken into account for the implementation and management of vector control programs in Africa.     

Reduction in Malaria Incidence following Indoor Residual Spraying with Actellic 300 CS in a Setting with Pyrethroid Resistance: Mutasa District,Zimbabwe

Background

More than half of malaria cases in Zimbabwe are concentrated in Manicaland Province, where seasonal malaria epidemics occur despite intensified control strategies. Recently, high levels of pyrethroid and carbamate resistance were detected in Anopheles funestus, the major malaria vector in eastern Zimbabwe. In response, a single round of indoor residual spraying (IRS) using pirimiphos-methyl (an organophosphate) was implemented in four high burden districts of Manicaland Province from November 1, 2014 to December 19, 2014. The objective of this study was to evaluate the effect of this programmatic switch in insecticides on malaria morbidity reported from health care facilities in Mutasa District, one of the worst affected districts in Manicaland Province.

Methods

The number of weekly malaria cases for each health facility 24 months prior to the 2014 IRS campaign and in the subsequent high transmission season were obtained from passive case surveillance. Environmental variables were extracted from remote-sensing data sources and linked to each health care facility. Negative binomial regression was used to model the weekly number of malaria cases, adjusted for seasonality and environmental variables.

Results

From December 2012 to May 2015, 124,206 malaria cases were reported from 42 health care facilities in Mutasa District. Based on a higher burden of malaria, 20 out of 31 municipal wards were sprayed in the district. Overall, 87.3% of target structures were sprayed and 92.1% of the target population protected. During the 6 months after the 2014 IRS campaign, a period when transmission would have otherwise peaked, the incidence of malaria was 38% lower than the preceding 24 months at health facilities in the sprayed wards.

Conclusions

Pirimiphos-methyl had a measurable impact on malaria incidence and is an effective insecticide for the control of An. funestus in eastern Zimbabwe.     

Changing patterns of malaria epidemiology between 2002 and 2010 in Western Kenya: the fall and rise of malaria

Background

The impact of insecticide treated nets (ITNs) on reducing malaria incidence is shown mainly through data collection from health facilities. Routine evaluation of long-term epidemiological and entomological dynamics is currently unavailable. In Kenya, new policies supporting the provision of free ITNs were implemented nationwide in June 2006. To evaluate the impacts of ITNs on malaria transmission, we conducted monthly surveys in three sentinel sites with different transmission intensities in western Kenya from 2002 to 2010.

Methods and Findings

Longitudinal samplings of malaria parasite prevalence in asymptomatic school children and vector abundance in randomly selected houses were undertaken monthly from February 2002. ITN ownership and usage surveys were conducted annually from 2004 to 2010. Asymptomatic malaria parasite prevalence and vector abundances gradually decreased in all three sites from 2002 to 2006, and parasite prevalence reached its lowest level from late 2006 to early 2007. The abundance of the major malaria vectors, Anopheles funestus and An. gambiae, increased about 5–10 folds in all study sites after 2007. However, the resurgence of vectors was highly variable between sites and species. By 2010, asymptomatic parasite prevalence in Kombewa had resurged to levels recorded in 2004/2005, but the resurgence was smaller in magnitude in the other sites. Household ITN ownership was at 50–70% in 2009, but the functional and effective bed net coverage in the population was estimated at 40.3%, 49.4% and 28.2% in 2010 in Iguhu, Kombewa, and Marani, respectively.

Conclusion

The resurgence in parasite prevalence and malaria vectors has been observed in two out of three sentinel sites in western Kenya despite a high ownership of ITNs. The likely factors contributing to malaria resurgence include reduced efficacy of ITNs, insecticide resistance in mosquitoes and lack of proper use of ITNs. These factors should be targeted to avoid further resurgence of malaria transmission.     

Identification and Characterisation of Aedes aegypti Aldehyde Dehydrogenases Involved in Pyrethroid Metabolism

Background

Pyrethroid insecticides, especially permethrin and deltamethrin, have been used extensively worldwide for mosquito control. However, insecticide resistance can spread through a population very rapidly under strong selection pressure from insecticide use. The upregulation of aldehyde dehydrogenase (ALDH) has been reported upon pyrethroid treatment. In Aedes aegypti, the increase in ALDH activity against the hydrolytic product of pyrethroid has been observed in DDT/permethrin-resistant strains. The objective of this study was to identify the role of individual ALDHs involved in pyrethroid metabolism.

Methodology/Principal Findings

Three ALDHs were identified; two of these, ALDH9948 and ALDH14080, were upregulated in terms of both mRNA and protein levels in a DDT/pyrethroid-resistant strain of Ae. aegypti. Recombinant ALDH9948 and ALDH14080 exhibited oxidase activities to catalyse the oxidation of a permethrin intermediate, phenoxybenzyl aldehyde (PBald), to phenoxybenzoic acid (PBacid).

Conclusions/Significance

ALDHs have been identified in association with permethrin resistance in Ae. aegypti. Characterisation of recombinant ALDHs confirmed the role of this protein in pyrethroid metabolism. Understanding the biochemical and molecular mechanisms of pyrethroid resistance provides information for improving vector control strategies.     

Synergy between Repellents and Organophosphates on Bed Nets: Efficacy and Behavioural Response of Natural Free-Flying An. gambiae Mosquitoes

Background

Chemicals are used on bed nets in order to prevent infected bites and to kill aggressive malaria vectors. Because pyrethroid resistance has become widespread in the main malaria vectors, research for alternative active ingredients becomes urgent. Mixing a repellent and a non-pyrethroid insecticide seemed to be a promising tool as mixtures in the laboratory showed the same features as pyrethroids.

Methodology/Principal Findings

We present here the results of two trials run against free-flying Anopheles gambiae populations comparing the effects of two insect repellents (either DEET or KBR 3023, also known as icaridin) and an organophosphate insecticide at low-doses (pirimiphos-methyl, PM) used alone and in combination on bed nets. We showed that mixtures of PM and the repellents induced higher exophily, blood feeding inhibition and mortality among wild susceptible and resistant malaria vectors than compounds used alone. Nevertheless the synergistic interactions are only involved in the high mortality induced by the two mixtures.

Conclusion

These field trials argue in favour of the strategy of mixing repellent and organophosphate on bed nets to better control resistant malaria vectors.     

Pyrethroid Resistance in Anopheles gambiae,in Bomi County,Liberia, Compromises Malaria Vector Control

Background

Long Lasting Insecticidal Nets (LLIN) and Indoor Residual Spraying (IRS) have both proven to be effective malaria vector control strategies in Africa and the new technology of insecticide treated durable wall lining (DL) is being evaluated. Sustaining these interventions at high coverage levels is logistically challenging and, furthermore, the increase in insecticide resistance in African malaria vectors may reduce the efficacy of these chemical based interventions. Monitoring of vector populations and evaluation of the efficacy of insecticide based control approaches should be integral components of malaria control programmes. This study reports on entomological survey conducted in 2011 in Bomi County, Liberia.

Methods

Anopheles gambiae larvae were collected from four sites in Bomi, Liberia, and reared in a field insectary. Two to five days old female adult An gambiae s.l. were tested using WHO tube (n = 2027) and cone (n = 580) bioassays in houses treated with DL or IRS. A sample of mosquitoes (n = 169) were identified to species/molecular form and screened for the presence of knock down resistance (kdr) alleles associated with pyrethroid resistance.

Results

Anopheles gambiae s.l tested were resistant to deltamethrin but fully susceptible to bendiocarb and fenithrothion. The corrected mortality of local mosquitoes exposed to houses treated with deltamethrin either via IRS or DL was 12% and 59% respectively, suggesting that resistance may affect the efficacy of these interventions. The presence of pyrethroid resistance was associated with a high frequency of the 1014F kdr allele (90.5%) although this mutation alone cannot explain the resistance levels observed.

Conclusion

High prevalence of resistance to deltamethrin in Bomi County may reduce the efficacy of malaria strategies relying on this class of insecticide. The findings highlight the urgent need to expand and sustain monitoring of insecticide resistance in Liberian malaria vectors, evaluate the effectiveness of existing interventions and develop appropriate resistance management strategies.     

Species Shifts in the Anopheles gambiae Complex: Do LLINs Successfully Control Anopheles arabiensis?

Introduction

High coverage of conventional and long-lasting insecticide treated nets (ITNs and LLINs) in parts of E Africa are associated with reductions in local malaria burdens. Shifts in malaria vector species ratio have coincided with the scale-up suggesting that some species are being controlled by ITNs/LLINs better than others.

Methods

Between 2005–2006 six experimental hut trials of ITNs and LLINs were conducted in parallel at two field stations in northeastern Tanzania; the first station was in Lower Moshi Rice Irrigation Zone, an area where An. arabiensis predominates, and the second was in coastal Muheza, where An. gambiae and An. funestus predominate. Five pyrethroids and one carbamate insecticide were evaluated on nets in terms of insecticide-induced mortality, blood-feeding inhibition and exiting rates.

Results

In the experimental hut trials mortality of An. arabiensis was consistently lower than that of An. gambiae and An. funestus. The mortality rates in trials with pyrethroid-treated nets ranged from 25–52% for An. arabiensis, 63–88% for An. gambiae s.s. and 53–78% for An. funestus. All pyrethroid-treated nets provided considerable protection for the occupants, despite being deliberately holed, with blood-feeding inhibition (percentage reduction in biting rates) being consistent between species. Veranda exiting rates did not differ between species. Percentage mortality of mosquitoes tested in cone bioassays on netting was similar for An. gambiae and An. arabiensis.

Conclusions

LLINs and ITNs treated with pyrethroids were more effective at killing An. gambiae and An. funestus than An. arabiensis. This could be a major contributing factor to the species shifts observed in East Africa following scale up of LLINs. With continued expansion of LLIN coverage in Africa An. arabiensis is likely to remain responsible for residual malaria transmission, and species shifts might be reported over larger areas. Supplementary control measures to LLINs may be necessary to control this vector species.     

Indoor residual spraying of insecticide and malaria morbidity in a high transmission intensity area of Uganda

Background

Recently the use of indoor residual spraying of insecticide (IRS) has greatly increased in Africa; however, limited data exist on the quantitative impacts of IRS on health outcomes in highly malaria endemic areas.

Methodology/Principal Findings

Routine data were collected on more than 90,000 patient visits at a single health facility over a 56 month period covering five rounds of IRS using three different insecticides. Temporal associations between the timing of IRS and the probability of a patient referred for microscopy having laboratory confirmed malaria were estimated controlling for seasonality and age. Considering patients less than five years of age there was a modest decrease in the odds of malaria following the 1^st round of IRS using DDT (OR = 0.76, p<0.001) and the 2^nd round using alpha-cypermethrin (OR = 0.83, p = 0.002). Following rounds 3–5 using bendiocarb there was a much greater decrease in the odds of malaria (ORs 0.34, 0.16, 0.17 respectively, p<0.001 for all comparisons). Overall, the impact of IRS was less pronounced among patients 5 years or older.

Conclusions/Significance

IRS was associated with a reduction in malaria morbidity in an area of high transmission intensity in Uganda and the benefits appeared to be greatest after switching to a carbamate class of insecticide.     

Plasma IP-10, apoptotic and angiogenic factors associated with fatal cerebral malaria in India

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.     

CYP6 P450 Enzymes and ACE-1 Duplication Produce Extreme and Multiple Insecticide Resistance in the Malaria Mosquito Anopheles gambiae

Malaria control relies heavily on pyrethroid insecticides, to which susceptibility is declining in Anopheles mosquitoes. To combat pyrethroid resistance, application of alternative insecticides is advocated for indoor residual spraying (IRS), and carbamates are increasingly important. Emergence of a very strong carbamate resistance phenotype in Anopheles gambiae from Tiassalé, Côte d''Ivoire, West Africa, is therefore a potentially major operational challenge, particularly because these malaria vectors now exhibit resistance to multiple insecticide classes. We investigated the genetic basis of resistance to the most commonly-applied carbamate, bendiocarb, in An. gambiae from Tiassalé. Geographically-replicated whole genome microarray experiments identified elevated P450 enzyme expression as associated with bendiocarb resistance, most notably genes from the CYP6 subfamily. P450s were further implicated in resistance phenotypes by induction of significantly elevated mortality to bendiocarb by the synergist piperonyl butoxide (PBO), which also enhanced the action of pyrethroids and an organophosphate. CYP6P3 and especially CYP6M2 produced bendiocarb resistance via transgenic expression in Drosophila in addition to pyrethroid resistance for both genes, and DDT resistance for CYP6M2 expression. CYP6M2 can thus cause resistance to three distinct classes of insecticide although the biochemical mechanism for carbamates is unclear because, in contrast to CYP6P3, recombinant CYP6M2 did not metabolise bendiocarb in vitro. Strongly bendiocarb resistant mosquitoes also displayed elevated expression of the acetylcholinesterase ACE-1 gene, arising at least in part from gene duplication, which confers a survival advantage to carriers of additional copies of resistant ACE-1 G119S alleles. Our results are alarming for vector-based malaria control. Extreme carbamate resistance in Tiassalé An. gambiae results from coupling of over-expressed target site allelic variants with heightened CYP6 P450 expression, which also provides resistance across contrasting insecticides. Mosquito populations displaying such a diverse basis of extreme and cross-resistance are likely to be unresponsive to standard insecticide resistance management practices.     

Synergy in Efficacy of Fungal Entomopathogens and Permethrin against West African Insecticide-Resistant Anopheles gambiae Mosquitoes

Background

Increasing incidences of insecticide resistance in malaria vectors are threatening the sustainable use of contemporary chemical vector control measures. Fungal entomopathogens provide a possible additional tool for the control of insecticide-resistant malaria mosquitoes. This study investigated the compatibility of the pyrethroid insecticide permethrin and two mosquito-pathogenic fungi, Beauveria bassiana and Metarhizium anisopliae, against a laboratory colony and field population of West African insecticide-resistant Anopheles gambiae s.s. mosquitoes.

Methodology/Findings

A range of fungus-insecticide combinations was used to test effects of timing and sequence of exposure. Both the laboratory-reared and field-collected mosquitoes were highly resistant to permethrin but susceptible to B. bassiana and M. anisopliae infection, inducing 100% mortality within nine days. Combinations of insecticide and fungus showed synergistic effects on mosquito survival. Fungal infection increased permethrin-induced mortality rates in wild An. gambiae s.s. mosquitoes and reciprocally, exposure to permethrin increased subsequent fungal-induced mortality rates in both colonies. Simultaneous co-exposure induced the highest mortality; up to 70.3±2% for a combined Beauveria and permethrin exposure within a time range of one gonotrophic cycle (4 days).

Conclusions/Significance

Combining fungi and permethrin induced a higher impact on mosquito survival than the use of these control agents alone. The observed synergism in efficacy shows the potential for integrated fungus-insecticide control measures to dramatically reduce malaria transmission and enable control at more moderate levels of coverage even in areas where insecticide resistance has rendered pyrethroids essentially ineffective.     

A New Long-Lasting Indoor Residual Formulation of the Organophosphate Insecticide Pirimiphos Methyl for Prolonged Control of Pyrethroid-Resistant Mosquitoes: An Experimental Hut Trial in Benin

Background

Indoor residual spraying (IRS) is widely used for malaria transmission control in sub-Saharan Africa. Resistance to pyrethroids in the mosquito Anopheles gambiae is a growing problem. There is an urgent need to develop long-lasting alternative insecticides to reduce selection pressure for pyrethroid resistance and to provide control with a single IRS application in countries with long transmission seasons.

Methods

Two capsule suspension formulations (CS) of the organophosphate pirimiphos methyl were evaluated as IRS treatments in experimental huts in an area of Benin where the mosquitoes Anopheles gambiae and Culex quinquefasciatus are resistant to pyrethroids but susceptible to organophosphates. The CS formulations were tested alongside an emulsifiable concentrate (EC) formulation of pirimiphos methyl and a CS formulation of the pyrethroid lambdacyhalothrin.

Results

The two CS formulations of pirimiphos methyl gave prolonged control of An. gambiae and Cx. quinquefasciatus. In cement huts application rates of 0.5 g/m² induced high mortality of An. gambiae for almost a year: overall mortality rates 87% (95% CI 82–91%) and 92% (95% CI 88–94%). In mud huts application rates of 1 g/m² induced high mortality of An. gambiae for 10 months: overall mortality rates 75% (95% CI 69–81%) and 76% (95% CI 68–83%). The EC formulation of pirimiphos methyl failed to control An. gambiae two months after spraying. The pyrethroid lambdacyhalothrin demonstrated prolonged residual activity in bioassay tests but failed to control pyrethroid resistant An. gambiae that entered the huts. Pirimiphos methyl CS was highly active against Culex quinquefasciatus and gave control for 10 months in cement huts and 6 months in mud huts.

Conclusion

Pirimiphos methyl CS (Actellic 300 CS) applied at 1 g/m² shows great promise for providing prolonged control of pyrethroid-resistant An gambiae and for delaying pyrethroid resistance. An alternative to DDT, giving year-round transmission control in sub-Saharan Africa is now a realistic prospect.