Evaluation of the Pfs25-IMX313/Matrix-M malaria transmission-blocking candidate vaccine in endemic settings.

Malaria control relies heavily on the use of anti-malarial drugs and insecticides against malaria parasites and mosquito vectors. Drug and insecticide resistance threatens the effectiveness of conventional malarial interventions; alternative control approaches are, therefore, needed. The development of malaria transmission-blocking vaccines that target the sexual stages in humans or mosquito vectors is among new approaches being pursued. Here, the immunological mechanisms underlying malaria transmission blocking, status of Pfs25-based vaccines are viewed, as well as approaches and capacity for first in-human evaluation of a transmission-blocking candidate vaccine Pfs25-IMX313/Matrix-M administered to semi-immune healthy individuals in endemic settings. It is concluded that institutions in low and middle income settings should be supported to conduct first-in human vaccine trials in order to stimulate innovative research and reduce the overdependence on developed countries for research and local interventions against many diseases of public health importance.

A differential expression of pyrethroid resistance genes in the malaria vector Anopheles funestus across Uganda is associated with patterns of gene flow.

BACKGROUND: Insecticide resistance is challenging the effectiveness of insecticide-based control interventions to reduce malaria burden in Africa. Understanding the molecular basis of insecticides resistance and patterns of gene flow in major malaria vectors such as Anopheles funestus are important steps for designing effective resistance management strategies. Here, we investigated the association between patterns of genetic structure and expression profiles of genes involved in the pyrethroid resistance in An. funestus across Uganda and neighboring Kenya. METHODS: Blood-fed mosquitoes An. funestus were collected across the four localities in Uganda and neighboring Kenya. A Microarray-based genome-wide transcription analysis was performed to identify the set of genes associated with permethrin resistance. 17 microsatellites markers were genotyped and used to establish patterns of genetic differentiation. RESULTS: Microarray-based genome-wide transcription profiling of pyrethroid resistance in four locations across Uganda (Arua, Bulambuli, Lira, and Tororo) and Kenya (Kisumu) revealed that resistance was mainly driven by metabolic resistance. The most commonly up-regulated genes in pyrethroid resistance mosquitoes include cytochrome P450s (CYP9K1, CYP6M7, CYP4H18, CYP4H17, CYP4C36). However, expression levels of key genes vary geographically such as the P450 CYP6M7 [Fold-change (FC) = 115.8 (Arua) vs 24.05 (Tororo) and 16.9 (Kisumu)]. In addition, several genes from other families were also over-expressed including Glutathione S-transferases (GSTs), carboxylesterases, trypsin, glycogenin, and nucleotide binding protein which probably contribute to insecticide resistance across Uganda and Kenya. Genotyping of 17 microsatellite loci in the five locations provided evidence that a geographical shift in the resistance mechanisms could be associated with patterns of population structure throughout East Africa. Genetic and population structure analyses indicated significant genetic differentiation between Arua and other localities (FST>0.03) and revealed a barrier to gene flow between Arua and other areas, possibly associated with Rift Valley. CONCLUSION: The correlation between patterns of genetic structure and variation in gene expression could be used to inform future interventions especially as new insecticides are gradually introduced.

Uptake of Plasmodium falciparum Gametocytes During Mosquito Bloodmeal by Direct and Membrane Feeding.

Plasmodium falciparum remains one of the leading causes of child mortality, and nearly half of the world's population is at risk of contracting malaria. While pathogenesis results from replication of asexual forms in human red blood cells, it is the sexually differentiated forms, gametocytes, which are responsible for the spread of the disease. For transmission to succeed, both mature male and female gametocytes must be taken up by a female Anopheles mosquito during its blood meal for subsequent differentiation into gametes and mating inside the mosquito gut. Observed circulating numbers of gametocytes in the human host are often surprisingly low. A pre-fertilization behavior, such as skin sequestration, has been hypothesized to explain the efficiency of human-to-mosquito transmission but has not been sufficiently tested due to a lack of appropriate tools. In this study, we describe the optimization of a qPCR tool that enables the relative quantification of gametocytes within very small input samples. Such a tool allows for the quantification of gametocytes in different compartments of the host and the vector that could potentially unravel mechanisms that enable highly efficient malaria transmission. We demonstrate the use of our gametocyte quantification method in mosquito blood meals from both direct skin feeding on Plasmodium gametocyte carriers and standard membrane feeding assay. Relative gametocyte abundance was not different between mosquitoes fed through a membrane or directly on the skin suggesting that there is no systematic enrichment of gametocytes picked up in the skin.

Establishing an international laboratory network for neglected tropical diseases: Understanding existing capacity in five WHO regions.

Background. Limited laboratory capacity is a significant bottleneck in meeting global targets for the control and elimination of neglected tropical diseases (NTD). Laboratories are essential for providing clinical data and monitoring data about the status and changes in NTD prevalence, and for detecting early drug resistance. Currently NTD laboratory networks are informal and specialist laboratory expertise is not well publicised, making it difficult to share global expertise and provide training, supervision, and quality assurance for NTD diagnosis and research. This study aimed to identify laboratories within five World Health Organisation regions (South-East Asia, Eastern Mediterranean, Americas, Western Pacific and Europe) that provide NTD services and could be regarded as national or regional reference laboratories, and to conduct a survey to document their networks and capacity to support NTD programmes. Methods. Potential NTD reference laboratories were identified through systematic searches, snowball sampling and key informants. Results. Thirty-two laboratories responded to the survey. The laboratories covered 17 different NTDs and their main regional and national roles were to provide technical support and training, research, test validation and standard setting. Two thirds of the laboratories were based in academic institutions and almost half had less than 11 staff. Although greater than 90 per cent of the laboratories had adequate technical skills to function as an NTD reference laboratory, almost all laboratories lacked systems for external verification that their results met international standards. Conclusions. This study highlights that although  many laboratories believed they could act as a reference laboratory, only a few had all the characteristics required to fulfil this role as they fell short in the standard and quality assurance of laboratory processes. Networks of high quality laboratories are essential for the control and elimination of disease and this study presents a critical first step in the development of such networks for NTDs.

Genome-Wide Transcription and Functional Analyses Reveal Heterogeneous Molecular Mechanisms Driving Pyrethroids Resistance in the Major Malaria Vector Anopheles funestus Across Africa.

Pyrethroid resistance in malaria vector, An. funestus is increasingly reported across Africa, threatening the sustainability of pyrethroid-based control interventions, including long lasting insecticidal nets (LLINs). Managing this problem requires understanding of the molecular basis of the resistance from different regions of the continent, to establish whether it is being driven by a single or independent selective events. Here, using a genome-wide transcription profiling of pyrethroid resistant populations from southern (Malawi), East (Uganda), and West Africa (Benin), we investigated the molecular basis of resistance, revealing strong differences between the different African regions. The duplicated cytochrome P450 genes (CYP6P9a and CYP6P9b) which were highly overexpressed in southern Africa are not the most upregulated in other regions, where other genes are more overexpressed, including GSTe2 in West (Benin) and CYP9K1 in East (Uganda). The lack of directional selection on both CYP6P9a and CYP6P9b in Uganda in contrast to southern Africa further supports the limited role of these genes outside southern Africa. However, other genes such as the P450 CYP9J11 are commonly overexpressed in all countries across Africa. Here, CYP9J11 is functionally characterized and shown to confer resistance to pyrethroids and moderate cross-resistance to carbamates (bendiocarb). The consistent overexpression of GSTe2 in Benin is coupled with a role of allelic variation at this gene as GAL4-UAS transgenic expression in Drosophila flies showed that the resistant 119F allele is highly efficient in conferring both DDT and permethrin resistance than the L119. The heterogeneity in the molecular basis of resistance and cross-resistance to insecticides in An. funestus populations throughout sub-Saharan African should be taken into account in designing resistance management strategies.

Widespread pyrethroid and DDT resistance in the major malaria vector Anopheles funestus in East Africa is driven by metabolic resistance mechanisms.

BACKGROUND: Establishing the extent, geographical distribution and mechanisms of insecticide resistance in malaria vectors is a prerequisite for resistance management. Here, we report a widespread distribution of insecticide resistance in the major malaria vector An. funestus across Uganda and western Kenya under the control of metabolic resistance mechanisms. METHODOLOGY/PRINCIPAL FINDINGS: Female An. funestus collected throughout Uganda and western Kenya exhibited a Plasmodium infection rate between 4.2 to 10.4%. Widespread resistance against both type I (permethrin) and II (deltamethrin) pyrethroids and DDT was observed across Uganda and western Kenya. All populations remain highly susceptible to carbamate, organophosphate and dieldrin insecticides. Knockdown resistance plays no role in the pyrethroid and DDT resistance as no kdr mutation associated with resistance was detected despite the presence of a F1021C replacement. Additionally, no signature of selection was observed on the sodium channel gene. Synergist assays and qRT-PCR indicated that metabolic resistance plays a major role notably through elevated expression of cytochrome P450s. DDT resistance mechanisms differ from West Africa as the L119F-GSTe2 mutation only explains a small proportion of the genetic variance to DDT resistance. CONCLUSION: The extensive distribution of pyrethroid and DDT resistance in East African An. funestus populations represents a challenge to the control of this vector. However, the observed carbamate and organophosphate susceptibility offers alternative solutions for resistance management.

Contrasting Plasmodium infection rates and insecticide susceptibility profiles between the sympatric sibling species Anopheles parensis and Anopheles funestus s.s: a potential challenge for malaria vector control in Uganda.

BACKGROUND: Although the An. funestus group conceals one of the major malaria vectors in Africa, little is known about the dynamics of members of this group across the continent. Here, we investigated the species composition, infection rate and susceptibility to insecticides of this species group in Uganda. METHODS: Indoor resting blood-fed Anopheles adult female mosquitoes were collected from 3 districts in Uganda. Mosquitoes morphologically belonging to the An. funestus group were identified to species by PCR. The sporozoite infection rates were determined by TaqMan and a nested PCR. Susceptibility to major insecticides was assessed using WHO bioassays. The potential role of four candidate resistance genes was assessed using qRT-PCR. RESULTS: An. funestus s.s. and An. parensis, were the only members of the An. funestus group identified. Both species were sympatric in Masindi (North-West), whereas only An. parensis was present in Mityana (Central) and Ntungamo (South-West). The Plasmodium falciparum infection detected in An. parensis (4.2%) by TaqMan could not be confirmed by nested PCR, whereas the 5.3% infection in An. funestus s.s. was confirmed. An. parensis was susceptible to most insecticides, however, a moderate resistance was observed against deltamethrin and DDT. In the sympatric population of Masindi, resistance was observed to pyrethroids (permethrin and deltamethrin) and DDT, but all the resistant mosquitoes belonged to An. funestus s.s. No significant over-expression was observed for the four P450 candidate genes CYP6M7, CYP9K1, CYP6P9 and CYP6AA4 between deltamethrin resistant and control An. parensis. However, when compared with the susceptible FANG An. funestus s.s strain, the CYP9K1 is significantly over-expressed in An. parensis (15-fold change; P < 0.001), suggesting it could play a role in the deltamethrin resistance. CONCLUSION: The contrasting infection rates and insecticide susceptibility profiles of both species highlights the importance of accurate species identification for successful vector control programs.