Multigene Phylogenetics Reveals Temporal Diversification of Major African Malaria Vectors

The major vectors of malaria in sub-Saharan Africa belong to subgenus Cellia. Yet, phylogenetic relationships and temporal diversification among African mosquito species have not been unambiguously determined. Knowledge about vector evolutionary history is crucial for correct interpretation of genetic changes identified through comparative genomics analyses. In this study, we estimated a molecular phylogeny using 49 gene sequences for the African malaria vectors An. gambiae, An. funestus, An. nili, the Asian malaria mosquito An. stephensi, and the outgroup species Culex quinquefasciatus and Aedes aegypti. To infer the phylogeny, we identified orthologous sequences uniformly distributed approximately every 5 Mb in the five chromosomal arms. The sequences were aligned and the phylogenetic trees were inferred using maximum likelihood and neighbor-joining methods. Bayesian molecular dating using a relaxed log normal model was used to infer divergence times. Trees from individual genes agreed with each other, placing An. nili as a basal clade that diversified from the studied malaria mosquito species 47.6 million years ago (mya). Other African malaria vectors originated more recently, and independently acquired traits related to vectorial capacity. The lineage leading to An. gambiae diverged 30.4 mya, while the African vector An. funestus and the Asian vector An. stephensi were the most closely related sister taxa that split 20.8 mya. These results were supported by consistently high bootstrap values in concatenated phylogenetic trees generated individually for each chromosomal arm. Genome-wide multigene phylogenetic analysis is a useful approach for discerning historic relationships among malaria vectors, providing a framework for the correct interpretation of genomic changes across species, and comprehending the evolutionary origins of this ubiquitous and deadly insect-borne disease.     

Inhibition of Malaria Infection in Transgenic Anopheline Mosquitoes Lacking Salivary Gland Cells

Malaria is an important global public health challenge, and is transmitted by anopheline mosquitoes during blood feeding. Mosquito vector control is one of the most effective methods to control malaria, and population replacement with genetically engineered mosquitoes to block its transmission is expected to become a new vector control strategy. The salivary glands are an effective target tissue for the expression of molecules that kill or inactivate malaria parasites. Moreover, salivary gland cells express a large number of molecules that facilitate blood feeding and parasite transmission to hosts. In the present study, we adapted a functional deficiency system in specific tissues by inducing cell death using the mouse Bcl-2-associated X protein (Bax) to the Asian malaria vector mosquito, Anopheles stephensi. We applied this technique to salivary gland cells, and produced a transgenic strain containing extremely low amounts of saliva. Although probing times for feeding on mice were longer in transgenic mosquitoes than in wild-type mosquitoes, transgenic mosquitoes still successfully ingested blood. Transgenic mosquitoes also exhibited a significant reduction in oocyst formation in the midgut in a rodent malaria model. These results indicate that mosquito saliva plays an important role in malaria infection in the midgut of anopheline mosquitoes. The dysfunction in the salivary glands enabled the inhibition of malaria transmission from hosts to mosquito midguts. Therefore, salivary components have potential in the development of new drugs or genetically engineered mosquitoes for malaria control.     

A New Malaria Agent in African Hominids

Plasmodium falciparum is the major human malaria agent responsible for 200 to 300 million infections and one to three million deaths annually, mainly among African infants. The origin and evolution of this pathogen within the human lineage is still unresolved. A single species, P. reichenowi, which infects chimpanzees, is known to be a close sister lineage of P. falciparum. Here we report the discovery of a new Plasmodium species infecting Hominids. This new species has been isolated in two chimpanzees (Pan troglodytes) kept as pets by villagers in Gabon (Africa). Analysis of its complete mitochondrial genome (5529 nucleotides including Cyt b, Cox I and Cox III genes) reveals an older divergence of this lineage from the clade that includes P. falciparum and P. reichenowi (∼21±9 Myrs ago using Bayesian methods and considering that the divergence between P. falciparum and P. reichenowi occurred 4 to 7 million years ago as generally considered in the literature). This time frame would be congruent with the radiation of hominoids, suggesting that this Plasmodium lineage might have been present in early hominoids and that they may both have experienced a simultaneous diversification. Investigation of the nuclear genome of this new species will further the understanding of the genetic adaptations of P. falciparum to humans. The risk of transfer and emergence of this new species in humans must be now seriously considered given that it was found in two chimpanzees living in contact with humans and its close relatedness to the most virulent agent of malaria.     

Protein Kinase C-Dependent Signaling Controls the Midgut Epithelial Barrier to Malaria Parasite Infection in Anopheline Mosquitoes

Anopheline mosquitoes are the primary vectors of parasites in the genus Plasmodium, the causative agents of malaria. Malaria parasites undergo a series of complex transformations upon ingestion by the mosquito host. During this process, the physical barrier of the midgut epithelium, along with innate immune defenses, functionally restrict parasite development. Although these defenses have been studied for some time, the regulatory factors that control them are poorly understood. The protein kinase C (PKC) gene family consists of serine/threonine kinases that serve as central signaling molecules and regulators of a broad spectrum of cellular processes including epithelial barrier function and immunity. Indeed, PKCs are highly conserved, ranging from 7 isoforms in Drosophila to 16 isoforms in mammals, yet none have been identified in mosquitoes. Despite conservation of the PKC gene family and their potential as targets for transmission-blocking strategies for malaria, no direct connections between PKCs, the mosquito immune response or epithelial barrier integrity are known. Here, we identify and characterize six PKC gene family members – PKCδ, PKCε, PKCζ, PKD, PKN, and an indeterminate conventional PKC − in Anopheles gambiae and Anopheles stephensi. Sequence and phylogenetic analyses of the anopheline PKCs support most subfamily assignments. All six PKCs are expressed in the midgut epithelia of A. gambiae and A. stephensi post-blood feeding, indicating availability for signaling in a tissue that is critical for malaria parasite development. Although inhibition of PKC enzymatic activity decreased NF-κB-regulated anti-microbial peptide expression in mosquito cells in vitro, PKC inhibition had no effect on expression of a panel of immune genes in the midgut epithelium in vivo. PKC inhibition did, however, significantly increase midgut barrier integrity and decrease development of P. falciparum oocysts in A. stephensi, suggesting that PKC-dependent signaling is a negative regulator of epithelial barrier function and a potential new target for transmission-blocking strategies.     

Ranking Malaria Risk Factors to Guide Malaria Control Efforts in African Highlands

Introduction

Malaria is re-emerging in most of the African highlands exposing the non immune population to deadly epidemics. A better understanding of the factors impacting transmission in the highlands is crucial to improve well targeted malaria control strategies.

Methods and Findings

A conceptual model of potential malaria risk factors in the highlands was built based on the available literature. Furthermore, the relative importance of these factors on malaria can be estimated through “classification and regression trees”, an unexploited statistical method in the malaria field. This CART method was used to analyse the malaria risk factors in the Burundi highlands. The results showed that Anopheles density was the best predictor for high malaria prevalence. Then lower rainfall, no vector control, higher minimum temperature and houses near breeding sites were associated by order of importance to higher Anopheles density.

Conclusions

In Burundi highlands monitoring Anopheles densities when rainfall is low may be able to predict epidemics. The conceptual model combined with the CART analysis is a decision support tool that could provide an important contribution toward the prevention and control of malaria by identifying major risk factors.     

Use of a Mixture Statistical Model in Studying Malaria Vectors Density

Vector control is a major step in the process of malaria control and elimination. This requires vector counts and appropriate statistical analyses of these counts. However, vector counts are often overdispersed. A non-parametric mixture of Poisson model (NPMP) is proposed to allow for overdispersion and better describe vector distribution. Mosquito collections using the Human Landing Catches as well as collection of environmental and climatic data were carried out from January to December 2009 in 28 villages in Southern Benin. A NPMP regression model with “village” as random effect is used to test statistical correlations between malaria vectors density and environmental and climatic factors. Furthermore, the villages were ranked using the latent classes derived from the NPMP model. Based on this classification of the villages, the impacts of four vector control strategies implemented in the villages were compared. Vector counts were highly variable and overdispersed with important proportion of zeros (75%). The NPMP model had a good aptitude to predict the observed values and showed that: i) proximity to freshwater body, market gardening, and high levels of rain were associated with high vector density; ii) water conveyance, cattle breeding, vegetation index were associated with low vector density. The 28 villages could then be ranked according to the mean vector number as estimated by the random part of the model after adjustment on all covariates. The NPMP model made it possible to describe the distribution of the vector across the study area. The villages were ranked according to the mean vector density after taking into account the most important covariates. This study demonstrates the necessity and possibility of adapting methods of vector counting and sampling to each setting.     

Malaria Vectors in Ecologically Heterogeneous Localities of the Colombian Pacific Region

The Colombian Pacific region is second nationally in number of malaria cases reported. This zone presents great ecological heterogeneity and Anopheles species diversity. However, little is known about the current spatial and temporal distribution of vector species. This study, conducted in three ecologically different localities of the Pacific region, aimed to evaluate the composition and distribution of Anopheles species and characterize transmission intensity. A total of 4,016 Anopheles mosquitoes were collected representing seven species. The composition and dominant species differed in each locality. Three species were infected with malaria parasites: Anopheles darlingi and An. calderoni were infected with Plasmodium falciparum and An. nuneztovari with Plasmodium vivax VK210 and VK247. Annual EIRs varied from 3.5–7.2 infective bites per year. These results confirm the importance of the primary vector An. nuneztovari in areas disturbed by human interventions, of An. darlingi in deforested margins of humid tropical rainforest and An. albimanus and the suspected vector An. calderoni in areas impacted by urbanization and large-scale palm oil agriculture close to the coast. This constitutes the first report in the Colombia Pacific region of naturally infected An. darlingi, and in Colombia of naturally infected An. calderoni. Further studies should evaluate the epidemiological importance of An. calderoni in the Pacific region.     

Patterns of Mitochondrial Variation within and between African Malaria Vectors, Anopheles Gambiae and An. Arabiensis, Suggest Extensive Gene Flow

Anopheles gambiae and An. arabiensis are mosquito species responsible for most malaria transmission in sub-Saharan Africa. They are also closely related sibling species that share chromosomal and molecular polymorphisms as a consequence of incomplete lineage sorting or introgressive hybridization. To help resolve these processes, this study examined the partitioning of mtDNA sequence variation within and between species across Africa, from both population genetic and phylogeographic perspectives. Based on partial gene sequences from the cytochrome b, ND1 and ND5 genes, haplotype diversity was high but sequences were very closely related. Within species, little or no population subdivision was detected, and there was no evidence for isolation by distance. Between species, there were no fixed nucleotide differences, a high proportion of shared polymorphisms, and eight haplotypes in common over distances as great as 6000 km. Only one of 16 shared polymorphisms led to an amino acid difference, and there was no compelling evidence for nonneutral variation. Parsimony networks constructed of haplotypes from both species revealed no correspondence of haplotype with either geography or taxonomy. This trend of low intraspecific genetic divergence is consistent with evidence from allozyme and microsatellite data and is interpreted in terms of both extensive gene flow and recent range expansion from relatively large, stable populations. We argue that retention of ancestral polymorphisms is a plausible but insufficient explanation for low interspecific genetic divergence, and that extensive hybridization is a contributing factor.     

Malaria epidemic early warning and detection in African highlands

Malaria epidemics have long been known to recur in the African highlands. Efforts to develop systems of early warning and detection for epidemics are outlined here with special emphasis on the Highland Malaria Project (HIMAL). This project has been conducting research on the operational implementation of a district-based surveillance and epidemic-monitoring system using a network of sentinel sites in four pilot districts of Kenya and Uganda. The potential use of weather monitoring as well as disease surveillance for effective early warning is being investigated.     

Asymptomatic Malaria in Refugees Living in a Non-Endemic South African City

Background

Asymptomatic malaria infection in refugees is both a threat to the lives of the individuals and the public in the host country. Although South Africa has been experiencing an unprecedented influx of refugees since 1994, data on malaria infection among refugees is lacking. Such information is critical since South Africa is among the countries that have planned to eliminate malaria. The objective of this study was to determine prevalence of asymptomatic malaria infection among a refugee population living in a city of KwaZulu-Natal province, South Africa.

Methods and Findings

A survey was conducted on adult refugee participants who attended a faith-based facility offering social services in a city of KwaZulu-Natal province, South Africa. The participants were screened for the presence of malaria using rapid diagnostic tests and microscopy. Demographic data for the participants were obtained using a closed ended questionnaire. Data was obtained for 303 participants consisting of 51.5% females and 47.5% males, ranging from 19 to 64 years old. More than 95% of them originated from sub-Saharan African countries. Two hundred and ninety participants provided a blood sample for screening of malaria. Of these, 3.8% tested positive for rapid diagnostic test and 5.9% for microscopy. The majority of malaria infections were due to Plasmodium falciparum.

Conclusions

The study confirms the presence of asymptomatic malaria infections among a refugee population residing in a city of KwaZulu-Natal province that is not endemic for malaria. The results have important implications for both public health and malaria control in South Africa, particularly since the country has decided to eliminate malaria by 2018. To achieve this goal, South Africa needs to expand research, surveillance and elimination activities to include non-endemic areas, particularly with high refugee populations. We further recommend use of powerful diagnostic tests such as PCR for these interventions.     

Mortality Patterns and Site Heterogeneity of Severe Malaria in African Children

Background

In this study we aimed to assess site heterogeneity of early, intermediate, and late mortality prediction in children with severe Plasmodium falciparum malaria in sub-Saharan Africa.

Methods

Medical records of 26,036 children admitted with severe Plasmodium falciparum malaria in six hospital research centers between December 2000 to May 2005 were analyzed. Demographic, clinical and laboratory data of children who died within 24 hours (early), between 24 and 47 hours (intermediate) and thereafter (48 hours or later, late mortality) were compared between groups and survivors.

Results

Overall mortality was 4·3% (N = 1,129). Median time to death varied across sites (P<0·001), ranging from 8h (3h–52h) in Lambaréné to 40h (10h–100h) in Kilifi. Fifty-eight percent of deaths occurred within 24 hours and intermediate and late mortality rate were 19% and 23%, respectively. Combining all sites, deep breathing, prostration and hypoglycemia were independent predictors for early, intermediate and late mortality (P<0·01). Site specific independent predictors for early death included prostration, coma and deep breathing at all sites (P<0·001). Site specific independent predictors for intermediate and late death largely varied between sites (P<0·001) and included between 1 and 7 different clinical and laboratory variables.

Conclusion

Site heterogeneity for mortality prediction is evident in African children with severe malaria. Prediction for early mortality has the highest consistency between sites.