Lisa J. Reimer

Relationship Between kdr Mutation and Resistance to Pyrethroid and DDT Insecticides in Natural Populations of Anopheles gambiae

Abstract The spread of insecticide resistance genes in Anopheles gambiae Giles sensu stricto threatens to compromise vector-based malaria control programs. Two mutations at the same locus in the voltage-gated sodium channel gene are known to confer knockdown resistance (kdr) to pyrethroids and DDT. Kdr-e involves a leucine-serine substitution, and it was until recently thought to be restricted to East Africa, whereas kdr-w, which involves a leucine-phenylalanine substitution, is associated with resistance in West Africa. In this study, we analyze the frequency and relationship between the kdr genotypes and resistance to type I and type II pyrethroids and DDT by using WHO test kits in both the Forest-M and S molecular forms of An. gambiae in Cameroon. Both kdr-w and kdr-e polymorphisms were found in sympatric An. gambiae, and in many cases in the same mosquito. Kdr-e and kdr-w were detected in both forms, but they were predominant in the S form. Both kdr-e and kdr-w were closely associated with resistance to DDT and weakly associated with resistance to type II pyrethroids. Kdr-w conferred greater resistance to permethrin than kdr-e. We also describe a modified diagnostic designed to detect both resistant alleles simultaneously.

An unusual distribution of the kdr gene among populations of Anopheles gambiae on the island of Bioko, Equatorial Guinea

In West Africa, Anopheles gambiae exists in discrete subpopulations known as the M and S molecular forms. Although these forms occur in sympatry, pyrethroid knock‐down resistance (kdr) is strongly associated with the S molecular form. On the island of Bioko, Equatorial Guinea we found high frequencies of the kdr mutation in M form individuals (55.8%) and a complete absence of kdr in the S form. We also report the absence of the kdr allele in M and S specimens from the harbour town of Tiko in Cameroon, representing the nearest continental population to Bioko. The kdr allele had previously been reported as absent in populations of An. gambiae on Bioko. Contrary to earlier reports, sequencing of intron‐1 of this sodium channel gene revealed no fixed differences between M form resistant and susceptible individuals. The mutation may have recently arisen independently in the M form on Bioko due to recent and intensive pyrethroid application.

Evidence for subdivision within the M molecular form of Anopheles gambiae

The principal vector of malaria in sub‐Saharan Africa, Anopheles gambiae is subdivided into two molecular forms M and S. Additionally, several chromosomal forms, characterized by the presence of various inversion polymorphisms, have been described. The molecular forms M and S each contain several chromosomal forms, including the Savanna, Mopti and Forest forms. The M and S molecular forms are now considered to be the reproductive units within A. gambiae and it has recently been argued that a low recombination rate in the centromeric region of the X chromosome has facilitated isolation between these forms. The status of the chromosomal forms remains unclear however. Therefore, we studied genetic differentiation between Savanna S, Forest S, Forest M and Mopti M populations using microsatellites. Genetic differentiation between Savanna S and Forest S populations is very low (FST = 0.0053 ± 0.0049), even across large distances. In comparison, the Mopti M and Forest M populations show a relatively high degree of genetic differentiation (FST = 0.0406 ± 0.0054) indicating that the M molecular form may not be a single entity, but could be subdivided into at least two distinct chromosomal forms. Previously it was proposed that inversions have played a role in the origin of species within the A. gambiae complex. We argue that a possible subdivision within the M molecular form could be understood through this process, with the acquisition of inversions leading to the expansion of the M molecular form into new habitat, dividing it into two distinct chromosomal forms.

Insecticidal bed nets and filariasis transmission in Papua New Guinea.

BACKGROUND Global efforts to eliminate lymphatic filariasis are based on the annual mass administration of antifilarial drugs to reduce the microfilaria reservoir available to the mosquito vector. Insecticide-treated bed nets are being widely used in areas in which filariasis and malaria are coendemic. METHODS We studied five villages in which five annual mass administrations of antifilarial drugs, which were completed in 1998, reduced the transmission of Wuchereria bancrofti, one of the nematodes that cause lymphatic filariasis. A total of 21,899 anopheles mosquitoes were collected for 26 months before and 11 to 36 months after bed nets treated with long-lasting insecticide were distributed in 2009. We evaluated the status of filarial infection and the presence of W. bancrofti DNA in anopheline mosquitoes before and after the introduction of insecticide-treated bed nets. We then used a model of population dynamics to estimate the probabilities of transmission cessation. RESULTS Village-specific rates of bites from anopheline mosquitoes ranged from 6.4 to 61.3 bites per person per day before the bed-net distribution and from 1.1 to 9.4 bites for 11 months after distribution (P<0.001). During the same period, the rate of detection of W. bancrofti in anopheline mosquitoes decreased from 1.8% to 0.4% (P=0.005), and the rate of detection of filarial DNA decreased from 19.4% to 14.9% (P=0.13). The annual transmission potential was 5 to 325 infective larvae inoculated per person per year before the bed-net distribution and 0 after the distribution. Among all five villages with a prevalence of microfilariae of 2 to 38%, the probability of transmission cessation increased from less than 1.0% before the bed-net distribution to a range of 4.9 to 95% in the 11 months after distribution. CONCLUSIONS Vector control with insecticide-treated bed nets is a valuable tool for W. bancrofti elimination in areas in which anopheline mosquitoes transmit the parasite. (Funded by the U.S. Public Health Service and the National Institutes of Health.).

The distribution of insecticide resistance in Anopheles gambiae s.l. populations from Cameroon: an update.

Insecticides are a key component of vector-based malaria control programmes in Cameroon. As part of ongoing resistance surveillance efforts, Anopheles gambiae s.l. female mosquitoes were exposed to organochlorine (DDT), a carbamate (bendiocarb), an organophosphate (malathion), and three pyrethroids (deltamethrin, lambda-cyhalothrin and permethrin) in WHO bioassay test kits. Results indicated a higher level of resistance (reduced mortality and knockdown effect) to DDT and pyrethroids in populations of A. gambiae s.s. than in A. arabiensis. The West and East African knockdown resistance (kdr) mutations were found in both species but at much higher frequencies in A. gambiae s.s. The West Africa kdr mutant was also more frequent in the A. gambiae S form than in the M form. No resistance to bendiocarb and malathion was found. Carbamate and organophosphorous compounds could thus be used as alternatives in locations in Cameroon where pyrethroid-resistant populations are found.

Tracing the Tiger: Population Genetics Provides Valuable Insights into the Aedes (Stegomyia) albopictus Invasion of the Australasian Region

Background The range of the Asian tiger mosquito Aedes albopictus is expanding globally, raising the threat of emerging and re-emerging arbovirus transmission risks including dengue and chikungunya. Its detection in Papua New Guineas (PNG) southern Fly River coastal region in 1988 and 1992 placed it 150 km from mainland Australia. However, it was not until 12 years later that it appeared on the Torres Strait Islands. We hypothesized that the extant PNG population expanded into the Torres Straits as an indirect effect of drought-proofing the southern Fly River coastal villages in response to El Nino-driven climate variability in the region (via the rollout of rainwater tanks and water storage containers). Methodology/Principal Findings Examination of the mosquitos mitochondrial DNA cytochrome oxidase I (COI) sequences and 13 novel nuclear microsatellites revealed evidence of substantial intermixing between PNGs southern Fly region and Torres Strait Island populations essentially compromising any island eradication attempts due to potential of reintroduction. However, two genetically distinct populations were identified in this region comprising the historically extant PNG populations and the exotic introduced population. Both COI sequence data and microsatellites showed the introduced population to have genetic affinities to populations from Timor Leste and Jakarta in the Indonesian region. Conclusions/Significance The Ae. albopictus invasion into the Australian region was not a range expansion out of PNG as suspected, but founded by other, genetically distinct population(s), with strong genetic affinities to populations sampled from the Indonesian region. We now suspect that the introduction of Ae. albopictus into the Australian region was driven by widespread illegal fishing activity originating from the Indonesian region during this period. Human sea traffic is apparently shuttling this mosquito between islands in the Torres Strait and the southern PNG mainland and this extensive movement may well compromise Ae. albopictus eradication attempts in this region.

Outbreak of Chikungunya Virus Infection, Vanimo, Papua New Guinea

In June 2012, health authorities in Papua New Guinea detected an increase in febrile illnesses in Vanimo. Chikungunya virus of the Eastern/Central/Southern African genotype harboring the E1:A226V mutation was identified. This ongoing outbreak has spread to ≥8 other provinces and has had a harmful effect on public health.

Barrier screens: a method to sample blood-fed and host-seeking exophilic mosquitoes

BackgroundDetermining the proportion of blood meals on humans by outdoor-feeding and resting mosquitoes is challenging. This is largely due to the difficulty of finding an adequate and unbiased sample of resting, engorged mosquitoes to enable the identification of host blood meal sources. This is particularly difficult in the south-west Pacific countries of Indonesia, the Solomon Islands and Papua New Guinea where thick vegetation constitutes the primary resting sites for the exophilic mosquitoes that are the primary malaria and filariasis vectors.MethodsBarrier screens of shade-cloth netting attached to bamboo poles were constructed between villages and likely areas where mosquitoes might seek blood meals or rest. Flying mosquitoes, obstructed by the barrier screens, would temporarily stop and could then be captured by aspiration at hourly intervals throughout the night.ResultsIn the three countries where this method was evaluated, blood-fed females of Anopheles farauti, Anopheles bancroftii, Anopheles longirostris, Anopheles sundaicus, Anopheles vagus, Anopheles kochi, Anopheles annularis, Anopheles tessellatus, Culex vishnui, Culex quinquefasciatus and Mansonia spp were collected while resting on the barrier screens. In addition, female Anopheles punctulatus and Armigeres spp as well as male An. farauti, Cx. vishnui, Cx. quinquefasciatus and Aedes species were similarly captured.ConclusionsBuilding barrier screens as temporary resting sites in areas where mosquitoes were likely to fly was an extremely time-effective method for collecting an unbiased representative sample of engorged mosquitoes for determining the human blood index.

Reduced Recombination Rate and Genetic Differentiation Between the M and S Forms of Anopheles gambiae s.s.

Genetic differentiation between the largely sympatric molecular forms M and S of Anopheles gambiae appears mostly limited to division 6 and part of division 5 of the X chromosome. This region is adjacent to the centromere and includes the rDNA that was used to define these forms. This localized differentiation between populations that experience gene flow strongly suggests that this region contains genes responsible for reproductive isolation. Regions adjacent to centromeres are known to experience less recombination in several species and it has recently been suggested that low recombination rates can facilitate the accumulation and maintenance of isolation genes in partially isolated populations. Therefore, we measured the recombination rate in division 5D/6 directly and estimate that it is at least 16-fold reduced across this region compared to the remainder of the X chromosome. Additionally, sequence data from four loci from field-collected mosquitoes from several West African countries show very strong differentiation between the molecular forms in division 5D/6, whereas none was observed in two loci elsewhere on the X chromosome. Furthermore, genetic variation was substantially lower in division 5D/6 compared to the two reference loci, and the inferred genealogies of the division 5D/6 genes show patterns consistent with selective sweeps. This suggests that the reduced recombination rate has increased the effect of selection on this region and that our data are consistent with the hypothesis that reduced recombination rates can play a role in the accumulation of isolation genes in the face of gene flow.

Quantitative analyses and modelling to support achievement of the 2020 goals for nine neglected tropical diseases

Quantitative analysis and mathematical models are useful tools in informing strategies to control or eliminate disease. Currently, there is an urgent need to develop these tools to inform policy to achieve the 2020 goals for neglected tropical diseases (NTDs). In this paper we give an overview of a collection of novel model-based analyses which aim to address key questions on the dynamics of transmission and control of nine NTDs: Chagas disease, visceral leishmaniasis, human African trypanosomiasis, leprosy, soil-transmitted helminths, schistosomiasis, lymphatic filariasis, onchocerciasis and trachoma. Several common themes resonate throughout these analyses, including: the importance of epidemiological setting on the success of interventions; targeting groups who are at highest risk of infection or re-infection; and reaching populations who are not accessing interventions and may act as a reservoir for infection,. The results also highlight the challenge of maintaining elimination ‘as a public health problem’ when true elimination is not reached. The models elucidate the factors that may be contributing most to persistence of disease and discuss the requirements for eventually achieving true elimination, if that is possible. Overall this collection presents new analyses to inform current control initiatives. These papers form a base from which further development of the models and more rigorous validation against a variety of datasets can help to give more detailed advice. At the moment, the models’ predictions are being considered as the world prepares for a final push towards control or elimination of neglected tropical diseases by 2020.