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We describe a novel one-step reverse transcriptase real-time PCR (direct RT-PCR) for Plasmodium falciparum malaria parasites that amplifies RNA targets directly from blood. We developed the assay to identify gametocyte-specific transcripts in parasites from patient blood samples, as a means of monitoring malaria parasite transmission in field settings. To perform the test, blood is added directly to a master mix in PCR tubes and analyzed by real-time PCR. The limit of detection of the assay on both conventional and portable real-time PCR instruments was 100 parasites/mL for 18S rRNA, and 1,000 parasites/mL for asexual (PFE0065W) and gametocyte (PF14_0367, PFGEXP5) mRNA targets. The usefulness of this assay in field studies was explored in samples from individuals living in a high-transmission region in Cameroon. The sensitivity and specificity of the assay compared with a standard two-step RT-PCR was 100% for 18S rRNA on both conventional and portable instruments. For PF14_0367, the sensitivity and specificity were 85.7% and 70.0%, respectively, on the conventional instrument and 78.6% and 90%, respectively, on the portable instrument. The concordance for assays run on the two instruments was 100% for 18S rRNA, and 79.2% for PF14_0367, with most discrepancies resulting from samples with low transcript levels. The results show asexual and sexual stage RNA targets can be detected directly from blood samples in a simple one-step test on a field-friendly instrument. This assay may be useful for monitoring malaria parasite transmission potential in elimination settings, where sensitive diagnostics are needed to evaluate the progress of malaria eradication initiatives.

Melanization is an immune response of mosquitoes that could potentially limit Plasmodium development. That mosquitoes rarely melanize Plasmodium falciparum in natural populations might result from immuno-suppression by the parasite, as has been observed in Aedes aegypti mosquitoes infected by Plasmodium gallinaceum. We tested this possibility in Anopheles gambiae mosquitoes infected by P. falciparum by comparing the ability to melanize a Sephadex bead of infected mosquitoes, of mosquitoes that had fed on infectious blood without becoming infected, and of control mosquitoes fed on uninfected blood. Rather than being immuno-suppressed, infected mosquitoes tended to have a stronger melanization response than mosquitoes in which the infection failed and than control mosquitoes, possibly because of immune activation after previous exposure to invading parasites. This finding suggests that P. falciparum relies on immune evasion rather than immuno-suppression to avoid being melanized and confirms that natural malaria transmission systems differ from laboratory models of mosquito-Plasmodium interactions.

We report the first finding of the knockdown Leu-Phe and Leu-Ser mutations associated with resistance to pyrethroids and DDT insecticides in the malaria mosquito Anopheles gambiae from Cameroon. The Leu-Phe mutation was found in both the M and S molecular forms of An. gambiae. Importantly, two specimens of the S molecular form were found to carry both mutations in a heterozygous state.

Anopheles arabiensis, together with Anopheles gambiae sensu stricto, are the most important vectors of human malaria in sub-Saharan Africa. The malaria situation keeps worsening, with 1 to 3 million deaths a year, and alternative strategies are needed to decrease malaria transmission intensity. In this paper, we studied the population structure of An. arabiensis from three sample sites on the remote Indian Ocean island of La Réunion. Our results showed strong genetic structuring between An. arabiensis populations on La Réunion, indicating the presence of barriers to gene flow. Reasons for such a high genetic differentiation are discussed, including the role of intensive control measures that have maintained reduced effective population size as well as a putative genetic adaptation to the environment. This strong structuring situation on the island represents an ideal framework for the prospect of genetic-based control trials.

The Anopheles gambiae genome sequence, together with the recent development of molecular tools for genome-wide analysis, promises new insights into the biology of the malaria vector. These insights should help define the best possible breakdown point for interrupting transmission in the mosquito vector. A survey of the intraspecific nucleotide diversity in coding regions of three different mosquito strains showed an average of one single nucleotide polymorphism (SNP) every 125 coding base pairs. High levels of nucleotide polymorphism were observed in mosquito immune-related genes and pathogen recognition receptors harbored higher replacement substitutions. Genotyping at SNP loci in natural populations of An. gambiae from three malaria foci showed contrasting patterns. The distribution of mutation Y443H in the thioester-containing protein 3 (TEP3) gene suggested this mutational event has occurred under selective constraints. Our results show that SNP-based studies will be valuable in identifying the sequence variation associated with phenotypic traits shaping vector competence.