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

Uptake of purines in <i>Plasmodium falciparum</i>-infected human erythrocytes is mostly mediated by the human Equilibrative Nucleoside Transporter and the human Facilitative Nucleobase Transporter

&lt;b&gt;Background&lt;/b&gt;: Plasmodium parasites are unable to synthesize purines de novo and have to salvage them from the host. Due to this limitation in the parasite, purine transporters have been an area of focus in the search for anti-malarial drugs. Although the uptake of purines through the human equilibrative nucleoside transporter (hENT1), the human facilitative nucleobase transporter (hFNT1) and the parasite-induced new permeation pathway (NPP) has been studied, no information appears to exist on the relative contribution of these three transporters to the uptake of adenosine and hypoxanthine. Using the appropriate transporter inhibitors, the role of each of these salvage pathways to the overall purine transport in intraerythrocytic Plasmodium falciparum was systematically investigated. &lt;b&gt;Methods&lt;/b&gt;: The transport of adenosine, hypoxanthine and adenine into uninfected and P. falciparum-infected human erythrocytes was investigated in the presence or absence of classical inhibitors of the hFNT1, hENT1 and NPP. The effective inhibition of the various transporters by the classical inhibitors was verified using appropriate known substrates. The ability of high concentration of unlabelled substrates to saturate these transporters was also studied. &lt;b&gt;Results&lt;/b&gt;: Transport of exogenous purine into infected or uninfected erythrocytes occurred primarily through saturable transporters rather than through the NPP. Hypoxanthine and adenine appeared to enter erythrocytes mainly through the hFNT1 nucleobase transporter whereas adenosine entered predominantly through the hENT1 nucleoside transporter. The rate of purine uptake was approximately doubled in infected cells compared to uninfected erythrocytes. In addition, it was found that the rate of adenosine uptake was considerably higher than the rate of hypoxanthine uptake in infected human red blood cells (RBC). It was also demonstrated that furosemide inhibited the transport of purine bases through hFNT1. &lt;b&gt;Conclusion&lt;/b&gt;: Collectively, the data obtained in this study clearly show that the endogenous host erythrocyte transporters hENT1 and hFNT1, rather than the NPP, are the major route of entry of purine into parasitized RBC. Inhibitors of hENT1 and hFNT1, as well as the NPP, should be considered in the development of anti-malarials targeted to purine transport

The impact of uniform and mixed species blood meals on the fitness of the mosquito vector Anopheles gambiae s.s: does a specialist pay for diversifying its host species diet?

We investigated the fitness consequences of specialization in an organism whose host choice has an immense impact on human health: the African malaria vector Anopheles gambiae s.s. We tested whether this mosquito’s specialism on humans can be attributed to the relative fitness benefits of specialist vs. generalist feeding strategies by contrasting their fecundity and survival on human-only and mixed host diets consisting of blood meals from humans and animals. When given only one blood meal, An. gambiae s.s. survived significantly longer on human and bovine blood, than on canine or avian blood. However, when blood fed repeatedly, there was no evidence that the fitness of An. gambiae s.s. fed a human-only diet was greater than those fed generalist diets. This suggests that the adoption of generalist host feeding strategies in An. gambiae s.s. is not constrained by intraspecific variation in the resource quality of blood from other available host species

Editorial: Special issue avian malaria

Avian malaria parasites or haemosporidia are found in bird species worldwide. This special issue focusses on three 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 host-parasite ecoevolutionary questions

Characterisation of species and diversity of Anopheles gambiae Keele Colony

Anopheles gambiae sensu stricto was recently reclassified as two species, An. coluzzii and An. gambiae s.s., in wild-caught mosquitoes, on the basis of the molecular form, denoted M or S, of a marker on the X chromosome. The An. gambiae Keele line is an outbred laboratory colony strain that was developed around 12 years ago by crosses between mosquitoes from 4 existing An. gambiae colonies. Laboratory colonies of mosquitoes often have limited genetic diversity because of small starting populations (founder effect) and subsequent fluctuations in colony size. Here we describe the characterisation of the chromosomal form(s) present in the Keele line, and investigate the diversity present in the colony using microsatellite markers on chromosome 3. We also characterise the large 2La inversion on chromosome 2. The results indicate that only the M-form of the chromosome X marker is present in the Keele colony, which was unexpected given that 3 of the 4 parent colonies were probably S-form. Levels of diversity were relatively high, as indicated by a mean number of microsatellite alleles of 6.25 across 4 microsatellites, in at least 25 mosquitoes. Both karyotypes of the inversion on chromosome 2 (2La/2L+a) were found to be present at approximately equal proportions. The Keele colony has a mixed M- and S-form origin, and in common with the PEST strain, we propose continuing to denote it as an An. gambiae s.s. line

An improved and highly sensitive microfluorimetric method for assessing susceptibility of Plasmodium falciparum to antimalarial drugs in vitro

BACKGROUND: The standard in vitro protocol currently in use for drug testing against Plasmodium falciparum, based on the incorporation of the purine [(3)H]-hypoxanthine, has two serious drawbacks. Firstly it is unsuitable for the testing of drugs that directly or indirectly impact on purine salvage or metabolism. Secondly, it relies on the use of expensive radiolabelled material, with added issues concerning detection, storage and waste disposal that make it unsuitable for use in many disease-endemic areas. Recently, the use of fluorochromes has been suggested as an alternative, but quenching of the fluorescence signal by the haemoglobin present in cultures of Plasmodium falciparum-infected erythrocytes severely limits the usefulness of this approach. METHODS: In order to resolve this problem, a new PicoGreen(®)-based procedure has been developed which incorporates additional steps to remove the interfering haemoglobin. The 50% inhibitory concentration (IC(50)) values of chloroquine and pyrimethamine against P. falciparum laboratory lines 3D7 and K1 were determined using the new protocol. RESULTS: The IC(50 )values of chloroquine and pyrimethamine against P. falciparum laboratory lines 3D7 and K1 determined with the new fluorescence-based protocol were statistically identical to those obtained using the traditional (3)H-hypoxanthine incorporation method, and consistent with literature values. CONCLUSION: The new method proved to be accurate, reproducible and sensitive, and has the advantage of being non-radioactive. The improved PicoGreen(® )method has the potential to replace traditional in vitro drug resistance assay techniques

Dielectric characterization of Plasmodium falciparum infected red blood cells using microfluidic impedance cytometry

Although malaria is the world’s most life-threatening parasitic disease, there is no clear understanding of how certain biophysical properties of infected cells change during the malaria infection cycle. In this article, we use microfluidic impedance cytometry to measure the dielectric properties of Plasmodium falciparum-infected red blood cells (i-RBCs) at specific time-points during the infection cycle. Individual parasites were identified within i-RBCs using Green Fluorescent Protein (GFP) emission. The dielectric properties of cell sub-populations were determined using the multi-shell model. Analysis showed that the membrane capacitance and cytoplasmic conductivity of i-RBCs increased along the infection time-course, due to membrane alterations caused by parasite infection. The volume ratio occupied by the parasite was estimated to vary from &lt;10% at earlier stages, to ~90% at later stages. This knowledge could be used to develop new label-free cell sorting techniques for sample pre-enrichment, improving diagnosis

Estimation of parasite age and synchrony status in Plasmodium falciparum infections

Human malaria parasites have complex but poorly understood population dynamics inside their human host. In some but not all infections, parasites progress synchronously through the 48 h lifecycle following erythrocyte invasion, such that at any one time there is a limited spread of parasites at a particular time (hours) post-invasion. Patients presenting with older parasites, and with asynchronous infections, have been reported to have higher risks of fatal outcomes, associated with higher parasite biomass and multiplication rates respectively. However, practical tools to assess synchrony and estimate parasite age post-invasion in patient samples are lacking. We have developed a novel method based on three genes differentially expressed over the parasite intra-erythrocytic lifecycle, and applied it to samples from patients with uncomplicated malaria attending two health clinics in Ghana. We found that most patients presented with synchronous infections, and with parasites within 12 h of erythrocyte invasion. Finally we investigated if clinical features such as fever and parasite density could act as predictors of parasite age and synchrony. The new method is a simple and practicable approach to study parasite dynamics in naturally-infected patients, and is a significant improvement on the subjective microscopical methods for parasite staging in vivo, aiding patient management

Susceptibility of Anopheles gambiae and Anopheles stephensi to tropical isolates of Plasmodium falciparum

Background: The susceptibility of anopheline mosquito species to Plasmodium infection is known to be variable with some mosquitoes more permissive to infection than others. Little work, however, has been carried out investigating the susceptibility of major malaria vectors to geographically diverse tropical isolates of Plasmodium falciparum aside from examining the possibility of infection extending its range from tropical regions into more temperate zones. Methods: This study investigates the susceptibility of two major tropical mosquito hosts (Anopheles gambiae and Anopheles stephensi) to P. falciparum isolates of different tropical geographical origins. Cultured parasite isolates were fed via membrane feeders simultaneously to both mosquito species and the resulting mosquito infections were compared. Results: Infection prevalence was variable with African parasites equally successful in both mosquito species, Thai parasites significantly more successful in An. stephensi, and PNG parasites largely unsuccessful in both species. Conclusion: Infection success of P. falciparum was variable according to geographical origin of both the parasite and the mosquito. Data presented raise the possibility that local adaptation of tropical parasites and mosquitoes has a role to play in limiting gene flow between allopatric parasite populations