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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.

This paper presents proof-of-principle of a miniatured low noise, low power, and high-sensitive malaria detection method based on the magnetoresistance (MR) sensor with a CMOS analog front-end (AFE) readout circuit for the detection of paramagnetic hemozoin particles. COMSOL Multiphysics¯ is employed for the finite-element (FEM) simulation of hemozoin particles to prove that the magnetic field generated from a multi-hemozoin particles system is within the sensing range of MR sensors. A CMOS AFE circuit is designed to convert the tiny current (approximately 60 μΑ) from MR sensors into a strong voltage signal able to be sampled and suppress high frequency and large amplitude noises stemming from the shift of the resultant magnetic field during the malaria diagnostic process. This CMOS AFE circuit is composed of a transimpedance amplifier (TIA) and a pair of Butterworth filters. This TIA can achieve a 98.5 dB dc gain and a 2.823 MHz bandwidth with low power consumption (375.65 μW) at a 3.3 V voltage supply and low input-referred noise (21.3857 nA/√Hz at 100 Hz). Butterworth filters can significantly reduce the high frequency and large amplitude noises caused by the unexpected shift of the magnetic field. The experimental results prove that the system provides an immediate response to samples with hemozoin particles and has the potential to achieve malaria parasite detection.

IntroductionThe use of reverse transcription, 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. A full understanding how the parasite adjusts its transmission in response to varying in-host environmental conditions during natural infections requires simultaneous quantification of early and late gametocytes. Here, we describe qRT-PCR assays that are specific for detection and quantification of early-stage gametocytes of P. falciparum.MethodsThe assays are based on expression of known early gametocyte genes (pfpeg4, pfg27, pfge1, pfge3 and pfgexp5). The specificity of the qRT-PCR assays was tested using purified stage II and stage V gametocytes. These validated assays were used with qRT-PCR assays targeting late stage (pfs25) and all-stage (pfs16) gametocyte-specific transcripts to quantify gametocytes in natural P. falciparum infections and in a controlled human clinical infection study.ResultsThe relative expression of pfpeg4, pfg27 and pfge3, but not of pfge1 and pfgexp5, was significantly higher in purified stage II compared to stage V gametocytes, indicating early gametocyte specificity. In natural infections, 71.2% of individuals had both early and late gametocyte transcripts (pfpeg4/pfg27 plus pfs25), 12.6% harboured only early gametocytes transcripts (pfpeg4/pfg27), and 15.2% had only late gametocytes transcripts (pfs25). In natural infections, the limit of detection was equivalent to 190 and 390 gametocytes/mL blood for pfpeg4 and pfg27, respectively. In infected volunteers, transcripts of pfpeg4 and pfg27 were detected shortly after the onset of blood stage infection, demonstrating the specificity of the assays.ConclusionThe pfpeg4 and pfg27 qRT-PCR assays can be used specifically to quantify circulating immature gametocytes. Quantification of early gametocytes will improve understanding of epidemiological processes that modulate P. falciparum transmission and enhance the evaluation of transmission blocking interventions.

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.

Background Anopheles gambiae mosquitoes are the primary vectors for the transmission of human malaria in Africa. Despite their importance from a global health perspective, we know very little about the regulatory elements controlling functional gene expression. Results We performed genome-wide profiling of chromatin accessibility by ATAC-seq in midguts and salivary glands of laboratory-reared An. gambiae mosquitoes experimentally infected with Plasmodium falciparum. Our results show that accessibility localized preferentially in promoters and in introns. The integration of ATAC-seq data with the transcriptome revealed by RNA-seq, and with ChIP-seq data, showed accessibility correlated positively both with gene expression, and with histone marks associated with active chromatin (H3K9ac/H3K27ac). The differential analysis of chromatin accessibility and the expression profiles of target genes between tissues allowed us to infer cis-regulatory elements controlling tissue specific gene expression, including functions linked to mosquito genes related to immune responses, like def1 or gam1. Additionally, the analysis of enriched motifs allowed us to predict the in vivo binding sites of tissue-specific transcription factors. The ATAC-seq assay also allowed the precise mapping of active regulatory regions, including TSS- and enhancer-like elements that annotate to genes with roles in malaria infection such as lrim1. Conclusion This study identifies genome-wide regulatory regions likely to play an essential function in the regulation of gene expression in An. gambiae. This study is important not only for advancing our understanding of the regulatory genome of the mosquito vector of human malaria, but also for possible application to the genetic manipulation of mosquitoes.

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 50-60 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 and other multigenic families. The temporary knockdown of PfAP2-O leads to a complete loss of var transcriptional memory and a decrease in cytoadherence. AP2-O-knocked down parasites exhibited also significant reductions in transmission through Anopheles mosquitoes. We propose that PfAP2-O is one of the major virulence gene transcriptional regulators and may, therefore, be exploited as an important target to disrupt severe malaria and block parasite transmission.

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.

Malaria is a serious infectious disease caused by unicellular eukaryotic parasites of the genus Plasmodium. Today more than 200 species exist [1], and whole-genome sequence data is available for around 22 species [2]. At least five different Plasmodium species can infect humans, of which Plasmodium falciparum is the major cause of morbidity and mortality worldwide [3]. Other species are specific to vertebrates including rodents, birds and lizards. The parasite moves between vertebrate hosts via the bite of a mosquito, which serves as the definitive host and vector. A parasite species is usually restricted in the number of different vertebrate host species it is able to...

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.

Background Multiple factors can influence stool sample integrity upon sample collection. Preservation of faecal samples for microbiome studies is therefore an important step, particularly in tropical regions where resources are limited and high temperatures may significantly influence microbiota profiles. Freezing is the accepted standard to preserve faecal samples however, cold chain methods are often unfeasible in fieldwork scenarios particularly in low and middle-income countries and alternatives are required. This study therefore aimed to address the impact of different preservative methods, time-to-freezing at ambient tropical temperatures, and stool heterogeneity on stool microbiome diversity and composition under real-life physical environments found in resource-limited fieldwork conditions. Methods Inner and outer stool samples collected from one specimen obtained from three children were stored using different storage preservation methods (raw, ethanol and RNAlater) in a Ugandan field setting. Mixed stool was also stored using these techniques and frozen at different time-to-freezing intervals post-collection from 0–32 h. Metataxonomic profiling was used to profile samples, targeting the V1–V2 regions of 16S rRNA with samples run on a MiSeq platform. Reads were trimmed, combined and aligned to the Greengenes database. Microbial diversity and composition data were generated and analysed using Quantitative Insights Into Microbial Ecology and R software. Results Child donor was the greatest predictor of microbiome variation between the stool samples, with all samples remaining identifiable to their child of origin despite the stool being stored under a variety of conditions. However, significant differences were observed in composition and diversity between preservation techniques, but intra-preservation technique variation was minimal for all preservation methods, and across the time-to-freezing range (0–32 h) used. Stool heterogeneity yielded no apparent microbiome differences. Conclusions Stool collected in a fieldwork setting for comparative microbiome analyses should ideally be stored as consistently as possible using the same preservation method throughout.

Introduction:\ud Despite the positive effects of intervention strategies that include insecticide-impregnated bed nets and artemisinin-based drug therapies, malaria still kills nearly 500,000 people per year and infects more than 200 million individuals globally. This, together with the emerging resistance of the parasite to frontline antimalarials, means that there is an urgent need for novel treatments that not only offer a cure for malaria but also prevent transmission. We show that by inhibiting an essential protein kinase that is a key regulator of RNA processing, we are able to kill the parasite in the blood and liver stages as well as prevent the development of the sexual-stage gametocytes, thereby blocking transmission to the mosquito.\ud \ud Rationale:\ud Our group has previously published a list of 36 protein kinases that are essential for blood-stage survival of the most virulent form of the human malaria parasite, Plasmodium falciparum. Here, we focused on one of these protein kinases from the P. falciparum CLK (cyclin-dependent–like kinase) family, PfCLK3, and reasoned that inhibition of this protein kinase by a small drug-like molecule would be effective at killing blood-stage parasites. We further hypothesized that because PfCLK3 plays a key role in RNA splicing, inhibition of this kinase would be effective at killing the parasite at all stages of the life cycle where RNA splicing is required. This would include blood, liver, and sexual stages.\ud \ud Results:\ud By screening a focused library of nearly 30,000 compounds, we identified a probe molecule that selectively inhibited PfCLK3 and killed blood-stage P. falciparum. Using a combination of evolved resistance and chemogenetics, we established that our probe molecule had parasiticidal activity by inhibition of PfCLK3. We further showed that inhibition of PfCLK3 in parasites resulted in a reduction in more than 400 gene transcripts known to be essential for parasite survival. The finding that the vast majority of the genes down-regulated by PfCLK3 inhibition contained introns supported the notion that inhibition of PfCLK3 killed the malaria parasite by preventing the splicing of essential parasite genes. Because there is a high degree of homology between orthologs of CLK3 in other Plasmodium species, it might be expected that our probe molecule would both inhibit CLK3 contained in other malaria parasite species and have effective parasiticidal activity in these parasites. This was indeed found to be the case, with our molecule showing potent inhibition of CLK3 from P. vivax and P. berghei as well as killing the blood stages of P. berghei and P. knowlesi. Furthermore, we demonstrated that CLK3 inhibition also kills liver-stage P. berghei parasites and prevents P. berghei infection in mice. Finally, we showed that inhibition of PfCLK3 prevents the development of P. falciparum gametocytes, thereby blocking the infection of mosquitoes.\ud \ud Conclusion:\ud We found that inhibition of the essential malaria protein kinase CLK3 can kill multiple species of malaria parasites at the blood stage as well as killing liver-stage parasites and blocking transmission of the parasite to mosquitoes by preventing gametocyte development. In this way, we validate Plasmodium spp. CLK3 as a target that can offer prophylactic, curative, and transmission-blocking potential.

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. Electronic supplementary material The online version of this article (10.1186/s12936-019-2719-9) contains supplementary material, which is available to authorized users.

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.

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. This study demonstrates proof-of-concept that near-infrared spectroscopy (NIRS) is capable of rapidly identifying laboratory strains of human malaria infection in African mosquito vectors. By using partial least square regression models based on malaria-infected and uninfected Anopheles gambiae mosquitoes, we showed that NIRS can detect oocyst- and sporozoite-stage Plasmodium falciparum infections with 88% and 95% accuracy, respectively. 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.

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 less than 10% at earlier stages, to approximately 90% at later stages. This knowledge could be used to develop new label-free cell sorting techniques for sample pre-enrichment, improving diagnosis.

The requirement for next-generation antimalarials to be both curative and transmission-blocking necessitates the identification of previously undiscovered druggable molecular pathways. We identified a selective inhibitor of the

Renal function is essential to maintain homeostasis. This is particularly significant for insects that undergo complete metamorphosis; larval mosquitoes must survive a freshwater habitat whereas adults are terrestrial, and mature females must maintain ion and fluid homeostasis after blood feeding. To investigate the physiological adaptations required for successful development to adulthood, we studied the Malpighian tubule transcriptome of Anopheles gambiae using Affymetrix arrays. We assessed transcription under several conditions; as third instar larvae, as adult males fed on sugar, as adult females fed on sugar, and adult females after a blood meal. In addition to providing the most detailed transcriptomic data to date on the Anopheles Malpighian tubules, the data provide unique information on the renal adaptations required for the switch from freshwater to terrestrial habitats, on gender differences, and on the contrast between nectar-feeding and haematophagy. We found clear differences associated with ontogenetic change in lifestyle, gender and diet, particularly in the neuropeptide receptors that control fluid secretion, and the water and ion transporters that impact volume and composition. These data were also combined with transcriptomics from the Drosophila melanogaster tubule, allowing meta-analysis of the genes which underpin tubule function across Diptera. To further investigate renal conservation across species we selected four D. melanogaster genes with orthologues highly enriched in the Anopheles tubules, and generated RNAi knockdown flies. Three of these genes proved essential, showing conservation of critical functions across 150 million years of phylogenetic separation. This extensive data-set is available as an online resource, MozTubules.org, and could potentially be mined for novel insecticide targets that can impact this critical organ in this pest species.

Blood is arguably the most important bodily fluid and its analysis provides crucial health status information. A first routine measure to narrow down diagnosis in clinical practice is the differential blood count, determining the frequency of all major blood cells. What is lacking to advance initial blood diagnostics is an unbiased and quick functional assessment of blood that can narrow down the diagnosis and generate specific hypotheses. To address this need, we introduce the continuous, cell-by-cell morpho-rheological (MORE) analysis of diluted whole blood, without labeling, enrichment or separation, at rates of 1000 cells/sec. In a drop of blood we can identify all major blood cells and characterize their pathological changes in several disease conditions in vitro and in patient samples. This approach takes previous results of mechanical studies on specifically isolated blood cells to the level of application directly in blood and adds a functional dimension to conventional blood analysis., eLife digest When you are sick and go to the doctor, it is often not obvious what exactly is wrong — what is causing fever, nausea, shortness of breath or other symptoms. It is important to find this out quickly so that the right action can be taken. One of the first steps is to obtain a blood sample and to count how many of the different blood cells are present in it. This is called a complete blood count, and the information it provides has turned out to be surprisingly useful. A large number of certain white blood cells, for example, can show that the body is fighting an infection. But there might be several reasons why the number of white blood cells has increased, so this information alone is often not enough for a specific diagnosis. There are many hundreds of possible tests that can supplement the results of a complete blood count. These might identify bacteria or measure the concentrations of certain molecules in the blood, for example. But which test will give the important clue that reveals the source of the illness? This can be difficult to predict. Although each test helps to narrow down the final diagnosis they become increasingly expensive and time-consuming to perform, and rapid action is often important when treating a disease. Can we get more decisive information from the initial blood test by measuring other properties of the blood cells? Toepfner et al. now show that this is possible using a technique called real-time deformability cytometry. This method forces the blood cells in a small drop of blood to flow extremely rapidly through a narrow microfluidic channel while they are imaged by a fast camera. A computer algorithm can then analyze the size and stiffness of the blood cells in real-time. Toepfner et al. show that this approach can detect characteristic changes that affect blood cells as a result of malaria, spherocytosis, bacterial and viral infections, and leukemia. Furthermore, many thousands of blood cells can be measured in a few minutes — fast enough to be suitable as a diagnostic test. These proof-of-concept findings can now be used to develop actual diagnostic tests for a wide range of blood-related diseases. The approach could also be used to test which of several drugs is working to treat a certain medical condition, and to monitor whether the treatment is progressing as planned.

V gamma 9V delta 2 T cells, the dominant gamma delta 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 V gamma 9V delta 2 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 V gamma 9V delta 2 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. Title in dissertation reference list: Vgamma9Vdelta2 T cells proliferate in response to phosphoantigens released from erythrocytes infected with Plasmodium falciparum at asexual and gametocyte stages

Blood is arguably the most important bodily fluid and its analysis provides crucial health status information. A first routine measure to narrow down diagnosis in clinical practice is the differential blood count, determining the frequency of all major blood cells. What is lacking to advance initial blood diagnostics is an unbiased and quick functional assessment of blood that can narrow down the diagnosis and generate specific hypotheses. To address this need, we introduce the continuous, cell-by-cell morpho-rheological (MORE) analysis of whole blood, without labeling, enrichment or separation, at rates of 1,000 cells/sec. In a drop of blood we can identify all major blood cells and characterize their pathological changes in several disease conditions in vitro and in patient samples. This approach takes previous results of mechanical studies on specifically isolated blood cells to the level of application directly in whole blood and adds a functional dimension to conventional blood analysis.

In this post-hoc analysis, we determined the influence of single nucleotide polymorphisms in host candidate immune genes on the outcome of drug resistant malaria in Cameroon.Human DNA from 760 patients from a previous clinical trial was subjected to mass spectrometry-based single nucleotide polymorphism (SNP) genotyping. Allele frequencies of candidate immune genes were calculated for 62 SNPs on 17 human chromosomes for their possible involvement in clearance of drug-resistant parasites with the triple mutations of pfcrt76T, pfmdr86Y, and pfmdr1246Y (TY) and pfdhfr51I, pfdhfr59R, pfdhfr108N, and pfdhps437G (IRNG) which were determined by dotblot or PCR-restriction analysis. Differences in SNP frequencies and association analysis were carried out by comparing Chi-square odds ratios (ORs) and stratified by Mantel-Haenzel statistics. An adjusted P value (OR)0·0008 was considered significant.Post-treatment drug failure rates were amodiaquine (36·4%); sulpadoxine/pyrimethamine-amodiaquine combination (15·4%); and sulphadoxine/pyrimethamine (18·1%). SNPs in IL22, IL-4R1, and CD36 appeared to have been associated with clearance of resistant parasites [p = 0·017, OR (C allele):1·44, 95% CI (OR): 1·06-1·95]; [P = 0·014, OR = 1·31, 95% CI (OR): 1·07-1·83]; [P = 5·78×10(-5), OR = 0·27, 95%CI (OR): 0·13-0·54], respectively, with high fever (39°C for 48 hours) [IL-22, P = 0·01, OR = 1·5, 95% CI (OR): 1·8-2·1] and also in high frequency among the Fulani participants [P = 0·006, OR = 1·83, 95% CI (OR): 1·11-3·08)]. The CD36-1264 null allele was completely absent in the northern population.Independent association of SNPs in IL22 and IL-4 with clearance of amodiaquine- and sulphadoxine/pyrimethamine-resistant parasites did not reach statistical significance, but may suggest that not all drug-resistant mutants are adversely affected by the same immune-mediated mechanisms of clearance.

Despite the global efforts made in the fight against malaria, the disease is resurging. One of the main causes is the resistance that Anopheles mosquitoes, vectors of the disease, have developed to insecticides. Anopheles must survive for at least 10 days to possibly transmit malaria. Therefore, to evaluate and improve malaria vector control interventions, it is imperative to monitor and accurately estimate the age distribution of mosquito populations as well as their population sizes. Here, we demonstrate a machine-learning based approach that uses mid-infrared spectra of mosquitoes to characterise simultaneously both age and species identity of females of the African malaria vector species Anopheles gambiae and An. arabiensis, using laboratory colonies. Mid-infrared spectroscopy-based prediction of mosquito age structures was statistically indistinguishable from true modelled distributions. The accuracy of classifying mosquitoes by species was 82.6%. The method has a negligible cost per mosquito, does not require highly trained personnel, is rapid, and so can be easily applied in both laboratory and field settings. Our results indicate this method is a promising alternative to current mosquito species and age-grading approaches, with further improvements to accuracy and expansion for use with wild mosquito vectors possible through collection of larger mid-infrared spectroscopy data sets.

SUMMARYThe 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 inPlasmodium falciparumwhich 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 costlyin vitroandin vivomethods 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 inP. 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.

The efficiency of malaria parasite development within mosquito vectors (sporogony) is a critical determinant of transmission. Sporogony is thought to be controlled by environmental conditions and mosquito/parasite genetic factors, with minimal contribution from mosquito behaviour during the period of parasite development. We tested this assumption by investigating whether successful sporogony of Plasmodium falciparum parasites through to human-infectious transmission stages is influenced by the host species upon which infected mosquitoes feed. Studies were conducted on two major African vector species that generally are found to differ in their innate host preferences: Anopheles arabiensis and An. gambiae sensu stricto. We show that the proportion of vectors developing transmissible infections (sporozoites) was influenced by the source of host blood consumed during sporogony. The direction of this effect was associated with the innate host preference of vectors: higher sporozoite prevalences were generated in the usually human-specialist An. gambiae s.s. feeding on human compared to cow blood, whereas the more zoophilic An. arabiensis had significantly higher prevalences after feeding on cow blood. The potential epidemiological implications of these results are discussed.

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.

Sporozoites, the invasive form of malaria parasites transmitted by mosquitoes, are quiescent while in the insect salivary glands. Sporozoites only differentiate inside of the hepatocytes of the mammalian host. We show that sporozoite latency is an active process controlled by a eukaryotic initiation factor-2alpha (eIF2alpha) kinase (IK2) and a phosphatase. IK2 activity is dominant in salivary gland sporozoites, leading to an inhibition of translation and accumulation of stalled mRNAs into granules. When sporozoites are injected into the mammalian host, an eIF2alpha phosphatase removes the PO4 from eIF2alpha-P, and the repression of translation is alleviated to permit their transformation into liver stages. In IK2 knockout sporozoites, eIF2alpha is not phosphorylated and the parasites transform prematurely into liver stages and lose their infectivity. Thus, to complete their life cycle, Plasmodium sporozoites exploit the mechanism that regulates stress responses in eukaryotic cells.

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.

Malaria and trypanosomiasis are diseases which afflict millions and for which novel therapies are urgently required. We have tested two well-characterized cell-penetrating peptides (CPPs) for antiparasitic activity. One CPP, designated TP10, has broad-spectrum antiparasitic activity against Plasmodium falciparum , both blood and mosquito stages, and against blood-stage Trypanosoma brucei brucei .

PfPK7 is an orphan protein kinase of Plasmodium falciparum with maximal homology to MEK3/6 and to fungal protein kinase A proteins in its C-terminal and N-terminal regions, respectively. We showed previously that recombinant PfPK7 is active on various substrates but is unable to phosphorylate the Plasmodium falciparum mitogen-activated protein kinase homologues, suggesting that it is not a MEK functional homologue. Using a reverse genetics approach to investigate the function of this enzyme in live parasites, we now show that Pf PK7 − parasite clones display phenotypes at two stages of their life cycle: first, a decrease in the rate of asexual growth in erythrocytes associated with a lower number of daughter merozoites generated per schizont, and second, a dramatic reduction in the ability to produce oocysts in the mosquito vector. A normal asexual growth rate and the ability to produce oocysts are restored if a functional copy of the Pf PK7 gene is reintroduced into the Pf PK7 − parasites. Hence, PfPK7 is involved in a pathway that regulates parasite proliferation and development.

Human malaria is caused by infection with protozoan parasites of the genus Plasmodium. Four species of Plasmodium are well known to cause malaria in humans: P. falciparum, P. vivax, P. malariae, and P. ovale (with two subspecies, P. ovale curtisi and P. ovale wallikeri [1]). Recently, a fifth species has been added to the list: the simian malaria species Plasmodium knowlesi has been discovered to be a major cause of zoonotic human malaria in parts of Southeast Asia (2). The parasite species vary in the severity of disease they cause, as a result of differences in their biology, with the highest morbidity and mortality resulting from infection with P. falciparum. Correct identification of the infecting species is required to select appropriate and effective antimalarial drugs for treatment.

Osmiophilic bodies are membrane-bound vesicles, found predominantly in Plasmodium female gametocytes, that become progressively more abundant as the gametocyte reaches full maturity. These vesicles lie beneath the subpellicular membrane of the gametocyte, and the release of their contents into the parasitophorous vacuole has been postulated to aid in the escape of gametocytes from the erythrocyte after ingestion by the mosquito. Currently, the only protein known to be associated with osmiophilic bodies in Plasmodium falciparum is Pfg377, a gametocyte-specific protein expressed at the onset of osmiophilic body development. Here we show by targeted gene disruption that Pfg377 plays a fundamental role in the formation of these organelles, and that female gametocytes lacking the full complement of osmiophilic bodies are significantly less efficient both in vitro and in vivo in their emergence from the erythrocytes upon induction of gametogenesis, a process whose timing is critical for fertilization with the short-lived male gamete. This reduced efficiency of emergence explains the significant defect in oocyst formation in mosquitoes fed blood meals containing Pfg377-negative gametocytes, resulting in an almost complete blockade of infection.

The kinome of the human malaria parasite Plasmodium falciparum includes two genes encoding mitogen-activated protein kinase (MAPK) homologues, pfmap-1 and pfmap-2, but no clear orthologue of the MAPK kinase (MAPKK) family, raising the question of the mode of activation and function of the plasmodial MAPKs. Functional studies in the rodent malaria model Plasmodium berghei recently showed the map-2 gene to be dispensable for asexual growth and gametocytogenesis, but essential for male gametogenesis in the mosquito vector. Here, we demonstrate by using a reverse genetics approach that the map-2 gene is essential for completion of the asexual cycle of P. falciparum, an unexpected result in view of the non-essentiality of the orthologous gene for P. berghei erythrocytic schizogony. This validates Pfmap-2 as a potential target for chemotherapeutic intervention. In contrast, the other P. falciparum MAPK, Pfmap-1, is required neither for in vitro schizogony and gametocytogenesis in erythrocytes, nor for gametogenesis and sporogony in the mosquito vector. However, Pfmap-2 protein levels are elevated in pfmap-1(-) parasites, suggesting that Pfmap-1 fulfils an important function in asexual parasites that necessitates compensatory adaptation in parasites lacking this enzyme.

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 analyse using short read 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 analyse 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 recombination that modify 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. We describe a novel web application that allows these data to be explored in detail.

We have compared the ability of five Plasmodium falciparum microsatellites and three antigen-coding loci to differentiate recrudescence from reinfection. We used 133 pairs of P. falciparum-infected blood samples collected during in vivo drug efficacy trials from three sites in Kenya with different malaria endemicities. There were no significant differences between the marker subsets in their ability to discriminate recrudescences from new infections across the three sites. Overall, microsatellite loci revealed significantly higher expected heterozygosity and multiplicity of infection levels than antigen-coding loci. The mean expected heterozygosity across all loci in the three populations was significantly higher with microsatellites (0.70, 0.78 and 0.79) than antigen-coding loci (0.53, 0.60 and 0.62) for Mwea, Tiwi and Bondo areas, respectively. These observations can be explained by three non-exclusive hypotheses: (i) microsatellites are more polymorphic than antigenic loci; (ii) partially immune hosts remove certain parasites from infections on the basis of their antigenic alleles; and/or (iii) recombination occurs in vitro or in vivo with microsatellites.

Plasmodium spp. undergo a complex obligate developmental cycle within their invertebrate vectors that enables transmission between vertebrate hosts. This developmental cycle involves sexual reproduction and then asexual multiplication, separated by phases of invasion and colonization of distinct vector tissues. As with other stages in the Plasmodium life cycle, there is exquisite adaptation of the malaria parasite to its changing environment as it transforms within the blood of its vertebrate host, through the different tissues of its mosquito vector and onwards to infect a new vertebrate host. Despite the intricacies inherent in these successive transformations, malaria parasites remain staggeringly successful at disseminating through their vertebrate host populations.

The route taken by malaria ookinetes to cross the midgut wall of the mosquito vector has been (and continues to be) controversial. In this article, we attempt to reinterpret and integrate the apparently conflicting observations of the past using the recently proposed Time Bomb model of ookinete invasion as a unifying framework. We argue that parasite entry into the midgut epithelium is always intracellular, occurs at sites where multiple adjacent host cells converge, and that parasite exit from the midgut epithelium can occur by either an intracellular or intercellular route, depending on the manner in which invaded host cells are extruded from the midgut wall. We also propose a novel hypothesis, the Cellular Treadmill model, to explain the movement of ookinetes through multiple adjacent midgut cells.

Plasmodium falciparumgametocytes grownin vitrowere fed through membrane feeders to laboratory-rearedAnopheles stephensimosquitoes. Intact midguts, including entire bloodmeal contents, were removed between 24 and 48 h post-bloodfeeding. Giemsa-stained histological sections were prepared from the midguts and examined by light microscopy. Contrary to previous reports, ookinetes were clearly visible within midgut epithelial cells, demonstrating intracellular migration across the midgut wall. Ookinetes entered epithelial cells through the lateral apical membrane at sites where 3 adjacent cells converged. There was no evidence for the existence of a morphologically distinct group of epithelial cells preferentially invaded by ookinetes. However, ookinete penetration was associated with significant morphological changes to invaded cells, including differential staining, condensation and fragmentation of the nucleus, vacuolization, loss of microvilli and various degrees of extrusion into the midgut lumen. Epithelial cells completely separated from the midgut wall were found within the midgut lumen. These cells were associated with invading parasites suggesting that ookinete penetration resulted in complete ejection of invaded cells from the midgut wall. Small clusters of morphologically altered midgut cells and invading parasites spanning the membranes of adjacent abnormal epithelial cells were observed, consistent with intracellular movement of ookinetes between neighbouring midgut cells. Extruded epithelial cells were also observed rarely in uninfected midguts. Epithelial cell extrusion, therefore, may be a general mechanism of tissue repair through which damaged cells are removed from the midgut wall rather than a parasite-specific response. These observations demonstrate that human malaria parasite infection of mosquitoes is consistent with, and provides further support for, the Time Bomb model of ookinete invasion of the mosquito midgut epithelium previously proposed for rodent malaria parasites.

Transmission-blocking vaccines prevent the development of Plasmodium parasite within the mosquito vector, thereby thwarting the spread of malaria through a community. The gold standard for determining the efficacy of a transmission-blocking vaccine is the standard membrane feeding assay. This assay requires the dissection of mosquitoes and microscopic counting of oocysts present on the mosquito mid-gut, typically at 7-10 days p.i. Here we describe a real-time quantitative PCR assay that is rapid, target-specific and robust, with a sensitive detection threshold and which may be employed earlier p.i. than the standard membrane feeding assay and is applicable to preserved material. The real-time PCR assay utilises the LightCycler platform and SYBR Green I detection system to amplify 180 bp of the asexual form of the Plasmodium falciparum rRNA gene. It has a quantitative range of greater than four orders of magnitude and a detection threshold of 10 parasites. Validation experiments using a monoclonal antibody of known blocking activity revealed the real-time PCR assay to give equivalent results to the standard membrane feeding assay. In addition, the PCR assay can establish the effect of such a monoclonal antibody on the parasites' development within the oocyst and on the sporozoite (the transmissible stage) yield, providing a more pertinent assessment of transmission blocking activity than is possible by the standard membrane feeding assay. This assay may also be employed to monitor the sporogonic development of P. falciparum parasites within the mosquito vector.

Standard techniques for counting parasites are often time-consuming, difficult and inaccurate, and occasionally unpleasant. Real-time quantitative polymerase chain reaction has recently been applied to parasitology, specifically Plasmodium, Toxoplasma, Leishmania and Neospora. These techniques are truly quantitative, give results over a range of 6-7 orders of magnitude, are quick to perform and require no manipulations post-amplification. They can be used to count genome numbers and to study levels of gene expression. The advantages and limitations of existing thermocyclers and applicable detection systems are discussed here, and promising new developments are highlighted.

There is a need for a specific, sensitive, robust, and large-scale method for diagnosis of drug resistance genes in natural Plasmodium falciparum infections. Established polymerase chain reaction (PCR)-based methods may be compromised by the multiplicity of P. falciparum genotypes in natural infections. Here we adopt a dot-blot method to detect point mutations at nucleotide 323 (residue 108) in the P. falciparum dihydrofolate reductase (dhfr) gene using allele-specific oligonucleotide probes. Serine (Ser) or threonine (Thr) at this position are associated with sensitivity to pyrimethamine while asparagine (Asn) is associated with resistance. The method combines PCR amplification and hybridization of amplified products with radiolabeled allele-specific probes. This technique is specific and sensitive; it detects parasitemia of less than 100 parasites/microl of blood, and can identify a minority parasite genotype down to 1% in a mixture. Analysis of P. falciparum isolates from Sudan, of known response to pyrimethamine, has demonstrated the sensitivity and specificity of the method and its ability to detect multiple genotypes in single infections. Furthermore, it has confirmed the association between pyrimethamine responses and dhfr alleles. The method has been successfully extended for analysis of other point mutations in dhfr at residues 51 and 59, which are associated with a high level of pyrimethamine resistance.

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.

Thaithong, S., Ranford-Cartwright, L. C., Siripoon, N., Harnyuttanakorn, P., Kanchanakhan, N. S., Seugorn, A., Rungsihirunrat, K., Cravo, P. V. L., and Beale, G. H. 2001. Plasmodium falciparum: Gene mutations and amplification of dihydrofolate reductase genes in parasites grown in vitro in presence of pyrimethamine. Experimental Parasitology98, 59–70. Samples of three pyrimethamine-sensitive clones of Plasmodium falciparum were grown for periods of 22–46 weeks in media containing stepwise increases in pyrimethamine concentrations and were seen to develop up to 1000-fold increases in resistance to the drug. With clone T9/94RC17, the dihydrofolate reductase (DHFR) gene was sequenced from 10 uncloned populations and 29 pure clones, all having increased resistance to pyrimethamine, and these sequences were compared with the sequence of the original pyrimethamine-sensitive clone. No changes in amino acid sequence were found to have occurred. Some resistant clones obtained by this method were then examined by pulsed-field gel electrophoresis, and the results indicated that there had been an increase in the size of chromosome 4. This was confirmed by hybridization of Southern blots with a chromosome 4-specific probe, the vacuolar ATPase subunit B gene, and a probe to DHFR. Dot-blotting with an oligonucleotide probe to DHFR confirmed that there had been increases up to 44-fold in copy number of the DHFR gene in the resistant strains. Resistant clones obtained by this procedure were then grown in medium lacking pyrimethamine for a period of nearly 2 years, and reversion nearly to the level of pyrimethamine sensitivity of the original clone T9/94RC17 was found to occur after about 16 months. Correspondingly, the chromosome 4 of the reverted population reverted to a size like that of the original sensitive clone T9/94RC17. The procedure of growing parasites in stepwise increases of pyrimethamine concentration was repeated with two other pyrimethamine-sensitive clones: TM4CB8-2.2.3 and G112CB1.1. (The DHFR gene of these clones encodes serine at position 108, in place of threonine as in clone T9/94RC17, and it was thought that this difference might conceivably affect the rate of mutation to asparagine at this position). Clones TM4CB8-2.2.3 and G112CB1.1 also responded by developing gradually increased resistance to pyrimethamine. However, in clone TM4CB8-2.2.3 a single mutation from Ile to Met at position 164 in the DHFR gene sequence was identified, and in clone G112CB1.1 there was a single mutation from Ala to Ser at position 16, but no mutations at position 108 were obtained in any of the clones studied here. In addition, chromosome 4 of clone TM4CB8-2.2.3 increased in size, presumably due to amplification of the DHFR gene. No increase in size was seen in clone G112CB1.1. We conclude that whereas some mutations producing changes in the amino acid sequence of the DHFR molecule may occur occasionally in clones or populations of P. falciparum grown in vitro in the presence of pyrimethamine, amplification of the DHFR gene following adaptation to growth in medium containing pyrimethamine occurs as a regular feature. The bearing of these findings on the development of pyrimethamine-resistant forms of malaria parasites in endemic areas is discussed.

Genetic diversity of malaria parasites represents a major issue in understanding several aspects of malaria infection and disease. Genotyping of Plasmodium falciparum infections with polymerase chain reaction (PCR)-based methods has therefore been introduced in epidemiological studies. Polymorphic regions of the msp1, msp2 and glurp genes are the most frequently used markers for genotyping, but methods may differ. A multicentre study was therefore conducted to evaluate the comparability of results from different laboratories when the same samples were analysed. Analyses of laboratory-cloned lines revealed high specificity but varying sensitivity. Detection of low-density clones was hampered in multiclonal infections. Analyses of isolates from Tanzania and Papua New Guinea revealed similar positivity rates with the same allelic types identified. The number of alleles detected per isolate, however, varied systematically between the laboratories especially at high parasite densities. When the analyses were repeated within the laboratories, high agreement was found in getting positive or negative results but with a random variation in the number of alleles detected. The msp2 locus appeared to be the most informative single marker for analyses of multiplicity of infection. Genotyping by PCR is a powerful tool for studies on genetic diversity of P. falciparum but this study has revealed limitations in comparing results on multiplicity of infection derived from different laboratories and emphasizes the need for highly standardized laboratory protocols.

Malaria parasites are capable of modulating the diversion of resources from asexual growth to the production of stages infective to mosquitoes (gametocytes). Increased rates of gametocytogenesis appear to be a general response to stress, both naturally encountered and novel. We have previously reported earlier and greater gametocytogenesis in response to subcurative antimalarial chemotherapy in the rodent malaria, Plasmodium chabaudi, in vivo. Using an immunofluorescent assay to detect parasites that had invaded red blood cell monolayers, we demonstrate a 5-fold increase in gametocytogenesis in the human malaria, P. falciparum, in vitro, in response to treatment with the antimalarial drug chloroquine. In all clones used, gametocytogenesis increased with increasing inhibition of asexual growth by chloroquine. Furthermore, there were clone differences in the relationship between stress and gametocyte production, implying the response was genetically variable. This was not, however, associated with chloroquine resistance. The epidemiological significance of these results is discussed.

The polymerase chain reaction (PCR) is widely used to detect and identify low numbers of blood forms of different species of human malaria parasites [1]. The method has demonstrated a high prevalence of sub-microscopic asymptomatic Plasmodium falciparum infections among inhabitants of malaria-endemic countries [2–5]. Absence of patent parasites from the peripheral blood is often associated with sub-patent parasitaemias that can last for several months, even when the possibility of reinfection can be ruled out [5,6]. However, it is not known whether such sub-patent long lasting infections can produce sexual forms and thus be capable of infecting mosquitoes. Some studies have indicated that the minimum density of gametocytes capable of infecting mosquitoes can be remarkably low (reviewed by Carter and Gwadz [7]), and it has been demonstrated that sexual forms are infectious to mosquitoes even when they occur at levels not detectable by microscopy [8]. Here we describe a reverse transcriptase polymerase chain reaction (RT-PCR) for detection of gametocytes of P. falciparum. Unlike asexual parasites, the mature gametocytes of P. falciparum are metabolically relatively inactive [9]. However, almost immediately mature gametocytes are activated to undergo gametogenesis, either upon ingestion by a mosquito or upon stimulation in vitro, they enter a period of intense protein synthesis [10]. Among the proteins which are produced rapidly and in large amounts following such activation, is the zygote and ookinete surface protein Pfs25 [10]. This protein and, as we show here, its mRNA molecules are specific to the activated gametocyte and to the zygote and ookinete stages into which it transforms. Both Pfs25 and its mRNA are totally absent from the asexual stage parasites of P. falciparum. We have exploited this property of the mRNA of Pfs25 in the activated gametocytes of P. falciparum in or* Corresponding author. Tel.: +44-131-6508658; fax: +44131-6673210; e-mail: H.Babiker@ed.ac.uk.