Malaria parasite transmission depends on the successful transition of Plasmodium through discrete developmental stages in the lumen of the mosquito midgut. Like the human intestinal tract, the mosquito midgut contains a diverse microbial flora, which may compromise the ability of Plasmodium to establish infection. We have identified an Enterobacter bacterium isolated from wild mosquito populations in Zambia that renders the mosquito 99% resistant to infection with the human malaria parasite Plasmodium falciparum by interfering with parasite development prior to invasion of the midgut epithelium. Phenotypic analyses showed that the anti-Plasmodium mechanism requires small populations of replicating bacteria and is mediated through a mosquito-independent interaction with the malaria parasite. We show that this anti-Plasmodium effect is largely caused by bacterial generation of reactive oxygen species.

BackgroundThere has been rapid scale-up of malaria vector control in the last ten years. Both of the primary control strategies, long-lasting pyrethroid treated nets and indoor residual spraying, rely on the use of a limited number of insecticides. Insecticide resistance, as measured by bioassay, has rapidly increased in prevalence and has come to the forefront as an issue that needs to be addressed to maintain the sustainability of malaria control and the drive to elimination. Zambia's programme reported high levels of resistance to the insecticides it used in 2010, and, as a result, increased its investment in resistance monitoring to support informed resistance management decisions.Methodology/Principal FindingsA country-wide survey on insecticide resistance in Zambian malaria vectors was performed using WHO bioassays to detect resistant phenotypes. Molecular techniques were used to detect target-site mutations and microarray to detect metabolic resistance mechanisms. Anopheles gambiae s.s. was resistant to pyrethroids, DDT and carbamates, with potential organophosphate resistance in one population. The resistant phenotypes were conferred by both target-site and metabolic mechanisms. Anopheles funestus s.s. was largely resistant to pyrethroids and carbamates, with potential resistance to DDT in two locations. The resistant phenotypes were conferred by elevated levels of cytochrome p450s.Conclusions/SignificanceCurrently, the Zambia National Malaria Control Centre is using these results to inform their vector control strategy. The methods employed here can serve as a template to all malaria-endemic countries striving to create a sustainable insecticide resistance management plan.

Surveillance for drug-resistant parasites in human blood is a major effort in malaria control. Here we report contrasting antifolate resistance polymorphisms in Plasmodium falciparum when parasites in human blood were compared with parasites in Anopheles vector mosquitoes from sleeping huts in rural Zambia. DNA encoding P. falciparum dihydrofolate reductase (EC 1.5.1.3) was amplified by PCR with allele-specific restriction enzyme digestions. Markedly prevalent pyrimethamine-resistant mutants were evident in human P. falciparum infections-S108N (>90%), with N51I, C59R, and 108N+51I+59R triple mutants (30-80%). This resistance level may be from selection pressure due to decades of sulfadoxine/pyrimethamine use in the region. In contrast, cycloguanil-resistant mutants were detected in very low frequency in parasites from human blood samples-S108T (13%), with A16V and 108T+16V double mutants (∼4%). Surprisingly, pyrimethamine-resistant mutants were of very low prevalence (2-12%) in the midguts of Anopheles arabiensis vector mosquitoes, but cycloguanil-resistant mutants were highly prevalent-S108T (90%), with A16V and the 108T+16V double mutant (49-57%). Structural analysis of the dihydrofolate reductase by in silico modeling revealed a key difference in the enzyme within the NADPH binding pocket, predicting the S108N enzyme to have reduced stability but the S108T enzyme to have increased stability. We conclude that P. falciparum can bear highly host-specific drug-resistant polymorphisms, most likely reflecting different selective pressures found in humans and mosquitoes. Thus, it may be useful to sample both human and mosquito vector infections to accurately ascertain the epidemiological status of drug-resistant alleles.

BackgroundMalaria, caused by the parasite Plasmodium falciparum, is a significant source of morbidity and mortality in southern Zambia. In the Mapanza Chiefdom, where transmission is seasonal, Anopheles arabiensis is the dominant malaria vector. The ability to predict larval habitats can help focus control measures.MethodsA survey was conducted in March-April 2007, at the end of the rainy season, to identify and map locations of water pooling and the occurrence anopheline larval habitats; this was repeated in October 2007 at the end of the dry season and in March-April 2008 during the next rainy season. Logistic regression and generalized linear mixed modeling were applied to assess the predictive value of terrain-based landscape indices along with LandSat imagery to identify aquatic habitats and, especially, those with anopheline mosquito larvae.ResultsApproximately two hundred aquatic habitat sites were identified with 69 percent positive for anopheline mosquitoes. Nine species of anopheline mosquitoes were identified, of which, 19% were An. arabiensis. Terrain-based landscape indices combined with LandSat predicted sites with water, sites with anopheline mosquitoes and sites specifically with An. arabiensis. These models were especially successful at ruling out potential locations, but had limited ability in predicting which anopheline species inhabited aquatic sites. Terrain indices derived from 90 meter Shuttle Radar Topography Mission (SRTM) digital elevation data (DEM) were better at predicting water drainage patterns and characterizing the landscape than those derived from 30 m Advanced Spaceborne Thermal Emission and Reflection Radiometer (ASTER) DEM.ConclusionsThe low number of aquatic habitats available and the ability to locate the limited number of aquatic habitat locations for surveillance, especially those containing anopheline larvae, suggest that larval control maybe a cost-effective control measure in the fight against malaria in Zambia and other regions with seasonal transmission. This work shows that, in areas of seasonal malaria transmission, incorporating terrain-based landscape models to the planning stages of vector control allows for the exclusion of significant portions of landscape that would be unsuitable for water to accumulate and for mosquito larvae occupation. With increasing free availability of satellite imagery such as SRTM and LandSat, the development of satellite imagery-based prediction models is becoming more accessible to vector management coordinators.

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 programs 1,2,3,4,5 . 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 essential 6,7,8 . 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 km 2 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 protocol 9,10 . 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 extraction 9 .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 quality 11 , a PCR for identification of Anopheles gambiae sibling species 10 and a nested PCR for typing of Plasmodium falciparum infection 12.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. Video LinkThe video component of this article can be found at https://ww...