Special issue: avian malaria.

Avian malaria parasites or haemosporidia are found in bird species worldwide. This special issue focuses on 3 most commonly studied genera: Haemoproteus, Plasmodium and Leucocytozoon. Seven research articles and reviews are provided to illustrate the breadth of knowledge of the diversity of avian malaria parasites in different regional habitats and across bird species, and the use of avian haemosporidian systems to examine host­parasite eco-evolutionary questions.

Influx of diverse, drug resistant and transmissible Plasmodium falciparum into a malaria-free setting in Qatar.

BACKGROUND: Successful control programs have impeded local malaria transmission in almost all Gulf Cooperation Council (GCC) countries: Qatar, Bahrain, Kuwait, Oman, the United Arab Emirates (UAE) and Saudi Arabia. Nevertheless, a prodigious influx of imported malaria via migrant workers sustains the threat of local transmission. Here we examine the origin of imported malaria in Qatar, assess genetic diversity and the prevalence of drug resistance genes in imported Plasmodium falciparum, and finally, address the potential for the reintroduction of local transmission. METHODS: This study examined imported malaria cases reported in Qatar, between 2013 and 2016. We focused on P. falciparum infections and estimated both total parasite and gametocyte density, using qPCR and qRT-PCR, respectively. We also examined ten neutral microsatellites and four genes associated with drug resistance, Pfmrp1, Pfcrt, Pfmdr1, and Pfkelch13, to assess the genetic diversity of imported P. falciparum strains, and the potential for propagating drug resistance genotypes respectively. RESULTS: The majority of imported malaria cases were P. vivax, while P. falciparum and mixed species infections (P. falciparum / P. vivax) were less frequent. The primary origin of P. vivax infection was the Indian subcontinent, while P. falciparum was mostly presented by African expatriates. Imported P. falciparum strains were highly diverse, carrying multiple genotypes, and infections also presented with early- and late-stage gametocytes. We observed a high prevalence of mutations implicated in drug resistance among these strains, including novel SNPs in Pfkelch13. CONCLUSIONS: The influx of genetically diverse P. falciparum, with multiple drug resistance markers and a high capacity for gametocyte production, represents a threat for the reestablishment of drug-resistant malaria into GCC countries. This scenario highlights the impact of mass international migration on the reintroduction of malaria to areas with absent or limited local transmission.

Indels, structural variation, and recombination drive genomic diversity in Plasmodium falciparum.

The malaria parasite Plasmodium falciparum has a great capacity for evolutionary adaptation to evade host immunity and develop drug resistance. Current understanding of parasite evolution is impeded by the fact that a large fraction of the genome is either highly repetitive or highly variable and thus difficult to analyze using short-read sequencing technologies. Here, we describe a resource of deep sequencing data on parents and progeny from genetic crosses, which has enabled us to perform the first genome-wide, integrated analysis of SNP, indel and complex polymorphisms, using Mendelian error rates as an indicator of genotypic accuracy. These data reveal that indels are exceptionally abundant, being more common than SNPs and thus the dominant mode of polymorphism within the core genome. We use the high density of SNP and indel markers to analyze patterns of meiotic recombination, confirming a high rate of crossover events and providing the first estimates for the rate of non-crossover events and the length of conversion tracts. We observe several instances of meiotic recombination within copy number variants associated with drug resistance, demonstrating a mechanism whereby fitness costs associated with resistance mutations could be compensated and greater phenotypic plasticity could be acquired.

Detection of Plasmodium falciparum infected Anopheles gambiae using near-infrared spectroscopy.

BACKGROUND: Large-scale surveillance of mosquito populations is crucial to assess the intensity of vector-borne disease transmission and the impact of control interventions. However, there is a lack of accurate, cost-effective and high-throughput tools for mass-screening of vectors. METHODS: A total of 750 Anopheles gambiae (Keele strain) mosquitoes were fed Plasmodium falciparum NF54 gametocytes through standard membrane feeding assay (SMFA) and afterwards maintained in insectary conditions to allow for oocyst (8 days) and sporozoite development (14 days). Thereupon, each mosquito was scanned using near infra-red spectroscopy (NIRS) and processed by quantitative polymerase chain reaction (qPCR) to determine the presence of infection and infection load. The spectra collected were randomly assigned to either a training dataset, used to develop calibrations for predicting oocyst- or sporozoite-infection through partial least square regressions (PLS); or to a test dataset, used for validating the calibration's prediction accuracy. RESULTS: NIRS detected oocyst- and sporozoite-stage P. falciparum infections with 88% and 95% accuracy, respectively. This study demonstrates proof-of-concept that NIRS is capable of rapidly identifying laboratory strains of human malaria infection in African mosquito vectors. CONCLUSIONS: Accurate, low-cost, reagent-free screening of mosquito populations enabled by NIRS could revolutionize surveillance and elimination strategies for the most important human malaria parasite in its primary African vector species. Further research is needed to evaluate how the method performs in the field following adjustments in the training datasets to include data from wild-caught infected and uninfected mosquitoes.

Correction to: Detection of Plasmodium falciparum infected Anopheles gambiae using near-infrared spectroscopy.

Following publication of the original article [1], it was flagged that the name of the author Lisa Ranford-Cartwright had been (incorrectly) given as 'Lisa-Ranford Cartwright.

Vγ9Vδ2 T cells proliferate in response to phosphoantigens released from erythrocytes infected with asexual and gametocyte stage Plasmodium falciparum.

Vγ9Vδ2 T cells, the dominant γδ T cell subset in human peripheral blood, are stimulated by phosphoantigens, of which (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate, is produced in the apicoplast of malaria parasites. Cell-free media from synchronised Plasmodium falciparum asexual ring, trophozoite, and schizont stage-cultures of high purity as well as media from ruptured schizont cultures, all stimulated Vγ9Vδ2 T cell proliferation, as did media from pure gametocyte cultures, whereas media from uninfected erythrocytes cultures did not. The media from ruptured schizont cultures and all the asexual and gametocyte stage cultures contained only background iron levels, suggesting that all erythrocyte haemoglobin is consumed as the parasites develop and supporting that the phosphoantigens were released from intact parasitized erythrocytes. The Vγ9Vδ2 T cell-stimulating agent was not affected by freezing, thawing or heating but was sensitive to phosphatase treatment, confirming its phosphoantigen identity. In summary, phosphoantigens are released from parasitised erythrocytes at all developmental blood stages.

The role of asymptomatic P. falciparum parasitaemia in the evolution of antimalarial drug resistance in areas of seasonal transmission.

In areas with seasonal transmission, proper management of acute malaria cases that arise in the transmission season can markedly reduce the disease burden. However, asymptomatic carriage of Plasmodium falciparum sustains a long-lasting reservoir in the transmission-free dry season that seeds cyclical malaria outbreaks. Clinical trials targeting asymptomatic parasitaemia in the dry season failed to interrupt the malaria epidemics that follow annual rains. These asymptomatic infections tend to carry multiple-clones, capable of producing gametocytes and infecting Anopheles mosquitoes. Different clones within an infection fluctuate consistently, indicative of interaction between clones during the long course of asymptomatic carriage. However, the therapy-free environment that prevails in the dry season dis-advantages the drug resistant lineages and favors the wild-type parasites. This review highlights some biological and epidemiological characteristics of asymptomatic parasitaemia and calls for consideration of policies to diminish parasite exposure to drugs "therapy-free" and allow natural selection to curb drug resistance in the above setting.

Rare-cell enrichment by a rapid, label-free, ultrasonic isopycnic technique for medical diagnostics.

One significant challenge in medical diagnostics lies in the development of label-free methods to separate different cells within complex biological samples. Here we demonstrate a generic, low-power ultrasonic separation technique, able to enrich different cell types based upon their physical properties. For malaria, we differentiate between infected and non-infected red blood cells in a fingerprick-sized drop of blood. We are able to achieve an enrichment of circulating cells infected by the ring stage of the parasite over nonparasitized red blood cells by between two and three orders of magnitude in less than 3 seconds (enabling detection at parasitemia levels as low as 0.0005%). In a second example, we also show that our methods can be used to enrich different cell types, concentrating Trypanosoma in blood at very low levels of infection, on disposable, low-cost chips.

The impact of low erythrocyte density in human blood on the fitness and energetic reserves of the African malaria vector Anopheles gambiae.

BACKGROUND: Anaemia is a common health problem in the developing world. This condition is characterized by a reduction in erythrocyte density, primarily from malnutrition and/or infectious diseases such as malaria. As red blood cells are the primary source of protein for haematophagous mosquitoes, any reduction could impede the ability of mosquito vectors to transmit malaria by influencing their fitness or that of the parasites they transmit. The aim of this study was to determine the impact of differences in the density of red blood cells in human blood on malaria vector (Anopheles gambiae sensu stricto) fitness. The hypotheses tested are that mosquito vector energetic reserves and fitness are negatively influenced by reductions in the red cell density of host human blood meals commensurate with those expected from severe anaemia. METHODS: Mosquitoes (An. gambiae s.s.) were offered blood meals of different packed cell volume (PCV) of human blood consistent with those arising from severe anaemia (15%) and normal PCV (50%). Associations between mosquito energetic reserves (lipid, glucose and glycogen) and fitness measures (reproduction and survival) and blood meal PCV were investigated. RESULTS: The amount of protein that malaria vectors acquired from blood feeding (indexed by haematin excretion) was significantly reduced at low blood PCV. However, mosquitoes feeding on blood of low PCV had the same oviposition rates as those feeding on blood of normal PCV, and showed an increase in egg production of around 15%. The long-term survival of An. gambiae s.s was reduced after feeding on low PCV blood, but PCV had no significant impact on the proportion of mosquitoes surviving through the minimal period required to develop and transmit malaria parasites (estimated as 14 days post-blood feeding). The impact of blood PCV on the energetic reserves of mosquitoes was relatively minor. CONCLUSIONS: These results suggest that feeding on human hosts whose PCV has been depleted due to severe anaemia does not significantly reduce the fitness or transmission potential of malaria vectors, and indicates that mosquitoes may be able exploit resources for reproduction more efficiently from blood of low rather than normal PCV.

Genetic and genomic approaches for the discovery of parasite genes involved in antimalarial drug resistance.

The biggest threat to the war on malaria is the continued evolution of drug resistance by the parasite. Resistance to almost all currently available antimalarials now exists in Plasmodium falciparum which causes the most suffering among all human malaria parasites. Monitoring of antimalarial efficacy and the development and subsequent spread of resistance has become an important part in the treatment and control of malaria. With recent reports of reduced efficacy of artemisinin, the current recommended treatment for uncomplicated malaria, there is urgent need for better methods to recognize and monitor drug resistance for effective treatment. Molecular markers have become a welcome addition to complement the more laborious and costly in vitro and in vivo methods that have traditionally been used to monitor drug resistance. However, there are currently no molecular markers for resistance to some antimalarials. This review highlights the role of the various genetic and genomic approaches that have been used in identifying the molecular markers that underlie drug resistance in P. falciparum. These approaches include; candidate genes, genetic linkage and genome-wide association studies. We discuss the requirements and limitations of each approach and use various examples to illustrate their contributions in identifying genomic regions of the parasite associated with antimalarial drug responses.

Evolutionary race: Malaria evolves to evade sickle cell protection.

Sickle cell haemoglobin (HbS) confers protection, albeit incomplete, from severe malaria. A recent study by Band et al. in Nature on parasite genomic variation of severe malaria cases identifies parasite genomic regions with alleles associated with severe disease risk in HbS individuals. The protective effect of HbS depends therefore on parasite genotype.

Mosquito host-seeking diel rhythm and chemosensory gene expression is affected by age and Plasmodium stages.

Malaria parasites can affect vector-related behaviours, increasing transmission success. Using Anopheles gambiae and Plasmodium falciparum, we consider the effect of interaction between infection stage and vector age on diel locomotion in response to human odour and the expression of antennal chemosensory genes. We identified age-dependent behavioural diel compartmentalisation by uninfected females post-blood meal. Infection disrupts overall and diel activity patterns compared with age-matched controls. In this study, mosquitoes carrying transmissible sporozoites were more active, shifting activity periods which corresponded with human host availability, in response to human odour. Older, uninfected, blood-fed females displayed reduced activity during their peak host-seeking period in response to human odour. Age- and infection stage-specific changes in odour-mediated locomotion coincide with altered transcript abundance of select chemosensory genes suggesting a possible molecular mechanism regulating the behaviour. We hypothesize that vector-related behaviours of female mosquitoes are altered by infection stage and further modulated by the age post-blood meal of the vector. Findings may have important implications for malaria transmission and disease dynamics.

Bio-products from Serratia marcescens isolated from Ghanaian Anopheles gambiae reduce Plasmodium falciparum burden in vector mosquitoes.

Novel ideas for control of mosquito-borne disease include the use of bacterial symbionts to reduce transmission. Bacteria belonging to the family Enterobacteriaceae isolated from mosquito midgut have shown promise in limiting Plasmodium intensity in the Anopheles vector. However, the mechanism of interaction between bacteria and parasite remains unclear. This study aimed at screening bio-products of two bacteria candidates for their anti-Plasmodial effects on mosquito stages of P. falciparum. Enterobacter cloacae and Serratia marcescens were isolated from field-caught Anopheles gambiae s.l. Spent media from liquid cultures of these bacteria were filtered, lyophilized and dissolved in sterile phosphate buffered saline (PBS). The re-dissolved bacterial products were added to gametocytaemic blood meals and fed to An. gambiae mosquitoes via membrane feeders. Control groups were fed on infected blood with or without lyophilized LB medium. The effect of the products on the infection prevalence and intensity of P. falciparum in mosquitoes was assessed by dissecting mosquito midguts and counting oocysts 10-11 days post-infection. S. marcescens bio-products elicited significant reduction in the number of mosquitoes infected (P=4.02 x10-5) with P. falciparum and the oocyst intensity (P<2 x 10-16) than E. cloacae products (P>0.05 for both prevalence and intensity) compared to the control (lyophilized LB medium). These data support the use of bioproducts released by S. marcescens for malaria control based on transmission blocking in the vector.

Alternative splicing of the Anopheles gambiae Dscam gene in diverse Plasmodium falciparum infections.

BACKGROUND: In insects, including Anopheles mosquitoes, Dscam (Down syndrome cell adhesion molecule) appears to be involved in phagocytosis of pathogens, and shows pathogen-specific splice-form expression between divergent pathogen (or parasite) types (e.g. between bacteria and Plasmodium or between Plasmodium berghei and Plasmodium falciparum). Here, data are presented from the first study of Dscam expression in response to genetic diversity within a parasite species. METHODS: In independent field and laboratory studies, a measure of Dscam splice-form diversity was compared between mosquitoes fed on blood that was free of P. falciparum to mosquitoes exposed to either single or mixed genotype infections of P. falciparum. RESULTS: Significant increases in Anopheles gambiae Dscam (AgDscam) receptor diversity were observed in parasite-exposed mosquitoes, but only weak evidence that AgDscam diversity rises further upon exposure to mixed genotype parasite infections was found. Finally, a cluster of AgDscam exon 4 variants that become especially common during Plasmodium invasion was identified. CONCLUSIONS: While the data clearly indicate that AgDscam diversity increases with P. falciparum exposure, they do not suggest that AgDscam diversity rises further in response to increased parasite diversity.

Host candidate gene polymorphisms and clearance of drug-resistant Plasmodium falciparum parasites.

BACKGROUND: Resistance to anti-malarial drugs is a widespread problem for control programmes for this devastating disease. Molecular tests are available for many anti-malarial drugs and are useful tools for the surveillance of drug resistance. However, the correlation of treatment outcome and molecular tests with particular parasite markers is not perfect, due in part to individuals who are able to clear genotypically drug-resistant parasites. This study aimed to identify molecular markers in the human genome that correlate with the clearance of malaria parasites after drug treatment, despite the drug resistance profile of the protozoan as predicted by molecular approaches. METHODS: 3721 samples from five African countries, which were known to contain genotypically drug resistant parasites, were analysed. These parasites were collected from patients who subsequently failed to clear their infection following drug treatment, as expected, but also from patients who successfully cleared their infections with drug-resistant parasites. 67 human polymorphisms (SNPs) on 17 chromosomes were analysed using Sequenom's mass spectrometry iPLEX gold platform, to identify regions of the human genome, which contribute to enhanced clearance of drug resistant parasites. RESULTS: An analysis of all data from the five countries revealed significant associations between the phenotype of ability to clear drug-resistant Plasmodium falciparum infection and human immune response loci common to all populations. Overall, three SNPs showed a significant association with clearance of drug-resistant parasites with odds ratios of 0.76 for SNP rs2706384 (95% CI 0.71-0.92, P = 0.005), 0.66 for SNP rs1805015 (95% CI 0.45-0.97, P = 0.03), and 0.67 for SNP rs1128127 (95% CI 0.45-0.99, P = 0.05), after adjustment for possible confounding factors. The first two SNPs (rs2706384 and rs1805015) are within loci involved in pro-inflammatory (interferon-gamma) and anti-inflammatory (IL-4) cytokine responses. The third locus encodes a protein involved in the degradation of misfolded proteins within the endoplasmic reticulum, and its role, if any, in the clearance phenotype is unclear. CONCLUSIONS: The study showed significant association of three loci in the human genome with the ability of parasite to clear drug-resistant P. falciparum in samples taken from five countries distributed across sub-Saharan Africa. Both SNP rs2706384 and SNP1805015 have previously been reported to be associated with risk of malaria infection in African populations. The loci are involved in the Th1/Th2 balance, and the association of SNPs within these genes suggests a key role for antibody in the clearance of drug-resistant parasites. It is possible that patients able to clear drug-resistant infections have an enhanced ability to control parasite growth.

Rational deployment of antimalarial drugs in Africa: should first-line combination drugs be reserved for paediatric malaria cases?

Artemisinin-based combination therapy is exerting novel selective pressure upon populations of Plasmodium falciparum across Africa. Levels of resistance to non-artemisinin partner drugs differ among parasite populations, and so the artemisinins are not uniformly protected from developing resistance, already present in South East Asia. Here, we consider strategies for prolonging the period of high level efficacy of combination therapy for two particular endemicities common in Africa. Under high intensity transmission, two alternating first-line combinations, ideally with antagonistic selective effects on the parasite genome, are advocated for paediatric malaria cases. This leaves second-line and other therapies for adult cases, and for intermittent preventive therapy. The drug portfolio would be selected to protect the 'premier' combination regimen from selection for resistance, while maximising impact on severe disease and mortality in children. In endemic areas subject to low, seasonal transmission of Plasmodium falciparum, such a strategy may deliver little benefit, as children represent a minority of cases. Nevertheless, the deployment of other drug-based interventions in low transmission and highly seasonal areas, such as mass drug administration aimed to interrupt malaria transmission, or intermittent preventive therapy, does provide an opportunity to diversify drug pressure. We thus propose an integrated approach to drug deployment, which minimises direct selective pressure on parasite populations from any one drug component. This approach is suitable for qualitatively and quantitatively different burdens of malaria, and should be supported by a programme of routine surveillance for emerging resistance.

The regulatory genome of the malaria vector Anopheles gambiae: integrating chromatin accessibility and gene expression.

Anopheles gambiae mosquitoes are primary human malaria vectors, but we know very little about their mechanisms of transcriptional regulation. We profiled chromatin accessibility by the assay for transposase-accessible chromatin by sequencing (ATAC-seq) in laboratory-reared A. gambiae mosquitoes experimentally infected with the human malaria parasite Plasmodium falciparum. By integrating ATAC-seq, RNA-seq and ChIP-seq data, we showed a positive correlation between accessibility at promoters and introns, gene expression and active histone marks. By comparing expression and chromatin structure patterns in different tissues, we were able to infer cis-regulatory elements controlling tissue-specific gene expression and to predict the in vivo binding sites of relevant transcription factors. The ATAC-seq assay also allowed the precise mapping of active regulatory regions, including novel transcription start sites and enhancers that were annotated to mosquito immune-related genes. Not only is this study important for advancing our understanding of mechanisms of transcriptional regulation in the mosquito vector of human malaria, but the information we produced also has great potential for developing new mosquito-control and anti-malaria strategies.

The Transcription Factor PfAP2-O Influences Virulence Gene Transcription and Sexual Development in Plasmodium falciparum.

The human malaria parasite Plasmodium falciparum expresses variant PfEMP1 proteins on the infected erythrocyte, which function as ligands for endothelial receptors in capillary vessels, leading to erythrocyte sequestration and severe malaria. The factors that orchestrate the mono-allelic expression of the 45-90 PfEMP1-encoding var genes within each parasite genome are still not fully identified. Here, we show that the transcription factor PfAP2-O influences the transcription of var genes. The temporary knockdown of PfAP2-O leads to a complete loss of var transcriptional memory and a decrease in cytoadherence in CD36 adherent parasites. AP2-O-knocked-down parasites exhibited also significant reductions in transmission through Anopheles mosquitoes. We propose that PfAP2-O is, beside its role in transmission stages, also one of the virulence gene transcriptional regulators and may therefore be exploited as an important target to disrupt severe malaria and block parasite transmission.

Real-time PCR assays for detection and quantification of early P. falciparum gametocyte stages.

The use of quantitative qRT-PCR assays for detection and quantification of late gametocyte stages has revealed the high transmission capacity of the human malaria parasite, Plasmodium falciparum. To understand how the parasite adjusts its transmission in response to in-host environmental conditions including antimalarials requires simultaneous quantification of early and late gametocytes. Here, we describe qRT-PCR assays that specifically detect and quantify early-stage P. falciparum gametocytes. The assays are based on expression of known early and late gametocyte genes and were developed using purified stage II and stage V gametocytes and tested in natural and controlled human infections. Genes pfpeg4 and pfg27 are specifically expressed at significant levels in early gametocytes with a limit of quantification of 190 and 390 gametocytes/mL, respectively. In infected volunteers, transcripts of pfpeg4 and pfg27 were detected shortly after the onset of blood stage infection. In natural infections, both early (pfpeg4/pfg27) and late gametocyte transcripts (pfs25) were detected in 71.2% of individuals, only early gametocyte transcripts in 12.6%, and only late gametocyte transcripts in 15.2%. The pfpeg4/pfg27 qRT-PCR assays are sensitive and specific for quantification of circulating sexually committed ring stages/early gametocytes and can be used to increase our understanding of epidemiological processes that modulate P. falciparum transmission.

An essential role for the Plasmodium Nek-2 Nima-related protein kinase in the sexual development of malaria parasites.

The molecular control of cell division and development in malaria parasites is far from understood. We previously showed that a Plasmodium gametocyte-specific NIMA-related protein kinase, nek-4, is required for completion of meiosis in the ookinete, the motile form that develops from the zygote in the mosquito vector. Here, we show that another NIMA-related kinase, Pfnek-2, is also predominantly expressed in gametocytes, and that Pfnek-2 is an active enzyme displaying an in vitro substrate preference distinct from that of Pfnek-4. A functional nek-2 gene is required for transmission of both Plasmodium falciparum and the rodent malaria parasite Plasmodium berghei to the mosquito vector, which is explained by the observation that disruption of the nek-2 gene in P. berghei causes dysregulation of DNA replication during meiosis and blocks ookinete development. This has implications (i) in our understanding of sexual development of malaria parasites and (ii) in the context of control strategies aimed at interfering with malaria transmission.