An in vivo humanized model to study homing and sequestration of Plasmodium falciparum transmission stages in the bone marrow.

Introduction: Recent evidence suggests that the bone marrow (BM) plays a key role in the diffusion of P. falciparum malaria by providing a "niche" for the maturation of the parasite gametocytes, responsible for human-to-mosquito transmission. Suitable humanized in vivo models to study the mechanisms of the interplay between the parasite and the human BM components are still missing. Methods: We report a novel experimental system based on the infusion of immature P. falciparum gametocytes into immunocompromised mice carrying chimeric ectopic ossicles whose stromal and bone compartments derive from human osteoprogenitor cells. Results: We demonstrate that immature gametocytes home within minutes to the ossicles and reach the extravascular regions, where they are retained in contact with different human BM stromal cell types. Discussion: Our model represents a powerful tool to study BM function and the interplay essential for parasite transmission in P. falciparum malaria and can be extended to study other infections in which the human BM plays a role.

Impedance-Based Rapid Diagnostic Tool for Single Malaria Parasite Detection.

This paper presents a custom, low-cost electronic system specifically designed for rapid and quantitative detection of the malaria parasite in a blood sample. The system exploits the paramagnetic properties of malaria-infected red blood cells (iRBCs) for their magnetophoretic capture on the surface of a silicon chip. A lattice of nickel magnetic micro-concentrators embedded in a silicon substrate concentrates the iRBCs above coplanar gold microelectrodes separated by 3 µm for their detection through an impedance measurement. The sensor is designed for a differential operation to remove the large contribution given by the blood sample. The electronic readout automatically balances the sensor before each experiment and reaches a resolution of 15 ppm in the impedance measurement at 1 MHz allowing a limit of detection of 40 parasite/µl with a capture time of 10 minutes. For better reliability of the results, four sensors are acquired during the same experiment. We demonstrate that the realized platform can also detect a single infected cell in real experimental conditions, measuring human blood infected by Plasmodium falciparum malaria specie.

Gametocyte-specific and all-blood-stage transmission-blocking chemotypes discovered from high throughput screening on Plasmodium falciparum gametocytes.

Blocking Plasmodium falciparum human-to-mosquito transmission is essential for malaria elimination, nonetheless drugs killing the pathogenic asexual stages are generally inactive on the parasite transmissible stages, the gametocytes. Due to technical and biological limitations in high throughput screening of non-proliferative stages, the search for gametocyte-killing molecules so far tested one tenth the number of compounds screened on asexual stages. Here we overcome these limitations and rapidly screened around 120,000 compounds, using not purified, bioluminescent mature gametocytes. Orthogonal gametocyte assays, selectivity assays on human cells and asexual parasites, followed by compound clustering, brought to the identification of 84 hits, half of which are gametocyte selective and half with comparable activity against sexual and asexual parasites. We validated seven chemotypes, three of which are, to the best of our knowledge, novel. These molecules are able to inhibit male gametocyte exflagellation and block parasite transmission through the Anopheles mosquito vector in a standard membrane feeding assay. This work shows that interrogating a wide and diverse chemical space, with a streamlined gametocyte HTS and hit validation funnel, holds promise for the identification of dual stage and gametocyte-selective compounds to be developed into new generation of transmission blocking drugs for malaria elimination.

The Nitrobenzoxadiazole Derivative NBDHEX Behaves as Plasmodium falciparum Gametocyte Selective Inhibitor with Malaria Parasite Transmission Blocking Activity.

This work describes the activity of 6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)hexan-1-ol (NBDHEX) and of its newly identified carboxylic acid metabolite on the human malaria parasite Plasmodium falciparum. NBDHEX has been previously identified as a potent cytotoxic agent against murine and human cancer cells as well as towards the protozoan parasite Giardia duodenalis. We show here that NBDHEX is active in vitro against all blood stages of P. falciparum, with the rare feature of killing the parasite stages transmissible to mosquitoes, the gametocytes, with a 4-fold higher potency than that on the pathogenic asexual stages. This activity importantly translates into blocking parasite transmission through the Anopheles vector in mosquito experimental infections. A mass spectrometry analysis identified covalent NBDHEX modifications in specific cysteine residues of five gametocyte proteins, possibly associated with its antiparasitic effect. The carboxylic acid metabolite of NBDHEX retains the gametocyte preferential inhibitory activity of the parent compound, making this novel P. falciparum transmission-blocking chemotype at least as a new tool to uncover biological processes targetable by gametocyte selective drugs. Both NBDHEX and its carboxylic acid metabolite show very limited in vitro cytotoxicity on VERO cells. This result and previous evidence that NBDHEX shows an excellent in vivo safety profile in mice and is orally active against human cancer xenografts make these molecules potential starting points to develop new P. falciparum transmission-blocking agents, enriching the repertoire of drugs needed to eliminate malaria.

Transmission-blocking drugs for malaria elimination.

Preventing human-to-mosquito transmission of malaria parasites provides possible solutions to interrupt the malaria parasite life cycle for malaria elimination. The development of validated routine assays enabled the discovery of such transmission-blocking compounds. Currently, one development priority remains on combinations of dual-active compounds with equipotent activity against both the disease-causing asexual and transmissible, sexual erythrocytic stages. Additionally, transmission-blocking compounds that target gametocyte-specific biology could be used in combination with compounds against asexual parasites. In either case, preventing transmission will reduce the risk of reinfection and, if different processes are targeted, also curb the spread of drug resistance. Here, we provide an updated roadmap to the discovery and development of new antimalarials with transmission-blocking activity to guide drug discovery for malaria elimination.

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.

A Lab-On-chip Tool for Rapid, Quantitative, and Stage-selective Diagnosis of Malaria.

Malaria remains the most important mosquito-borne infectious disease worldwide, with 229 million new cases and 409.000 deaths in 2019. The infection is caused by a protozoan parasite which attacks red blood cells by feeding on hemoglobin and transforming it into hemozoin. Despite the WHO recommendation of prompt malaria diagnosis, the quality of microscopy-based diagnosis is frequently inadequate while rapid diagnostic tests based on antigens are not quantitative and still affected by non-negligible false negative/positive results. PCR-based methods are highly performant but still not widely used in endemic areas. Here, a diagnostic tool (TMek), based on the paramagnetic properties of hemozoin nanocrystals in infected red blood cells (i-RBCs), is reported on. Exploiting the competition between gravity and magnetic forces, i-RBCs in a whole blood specimen are sorted and electrically detected in a microchip. The amplitude and time evolution of the electrical signal allow for the quantification of i-RBCs (in the range 10-105 i-RBC µL-1) and the distinction of the infection stage. A preliminary validation study on 75 patients with clinical suspect of malaria shows on-field operability, without false negative and a few false positive results. These findings indicate the potential of TMek as a quantitative, stage-selective, rapid test for malaria.

Plasmodium falciparum sexual parasites regulate infected erythrocyte permeability.

To ensure the transport of nutrients necessary for their survival, Plasmodium falciparum parasites increase erythrocyte permeability to diverse solutes. These new permeation pathways (NPPs) have been extensively characterized in the pathogenic asexual parasite stages, however the existence of NPPs has never been investigated in gametocytes, the sexual stages responsible for transmission to mosquitoes. Here, we show that NPPs are still active in erythrocytes infected with immature gametocytes and that this activity declines along gametocyte maturation. Our results indicate that NPPs are regulated by cyclic AMP (cAMP) signaling cascade, and that the decrease in cAMP levels in mature stages results in a slowdown of NPP activity. We also show that NPPs facilitate the uptake of artemisinin derivatives and that phosphodiesterase (PDE) inhibitors can reactivate NPPs and increase drug uptake in mature gametocytes. These processes are predicted to play a key role in P. falciparum gametocyte biology and susceptibility to antimalarials.

Inhibition of Resistance-Refractory P. falciparum Kinase PKG Delivers Prophylactic, Blood Stage, and Transmission-Blocking Antiplasmodial Activity.

The search for antimalarial chemotypes with modes of action unrelated to existing drugs has intensified with the recent failure of first-line therapies across Southeast Asia. Here, we show that the trisubstituted imidazole MMV030084 potently inhibits hepatocyte invasion by Plasmodium sporozoites, merozoite egress from asexual blood stage schizonts, and male gamete exflagellation. Metabolomic, phosphoproteomic, and chemoproteomic studies, validated with conditional knockdown parasites, molecular docking, and recombinant kinase assays, identified cGMP-dependent protein kinase (PKG) as the primary target of MMV030084. PKG is known to play essential roles in Plasmodium invasion of and egress from host cells, matching MMV030084's activity profile. Resistance selections and gene editing identified tyrosine kinase-like protein 3 as a low-level resistance mediator for PKG inhibitors, while PKG itself never mutated under pressure. These studies highlight PKG as a resistance-refractory antimalarial target throughout the Plasmodium life cycle and promote MMV030084 as a promising Plasmodium PKG-targeting chemotype.

Critical Steps of Plasmodium falciparum Ookinete Maturation.

The egress and fertilization of Plasmodium gametes and development of a motile ookinete are the first crucial steps that mediate the successful transmission of the malaria parasites from humans to the Anopheles vector. However, limited information exists about the cell biology and regulation of this process. Technical impediments in the establishment of in vitro conditions for ookinete maturation in Plasmodium falciparum and other human malaria parasites further constrain a detailed characterization of ookinete maturation. Here, using fluorescence microscopy and immunolabeling, we compared P. falciparum ookinete maturation in Anopheles coluzzii mosquitoes in vivo and in cell culture in vitro. Our results identified two critical steps in ookinete maturation that are regulated by distinct mosquito factors, thereby highlighting the role of the mosquito environment in the transmission efficiency of malaria parasites.

Antimalarial activity of primaquine operates via a two-step biochemical relay.

Primaquine (PQ) is an essential antimalarial drug but despite being developed over 70 years ago, its mode of action is unclear. Here, we demonstrate that hydroxylated-PQ metabolites (OH-PQm) are responsible for efficacy against liver and sexual transmission stages of Plasmodium falciparum. The antimalarial activity of PQ against liver stages depends on host CYP2D6 status, whilst OH-PQm display direct, CYP2D6-independent, activity. PQ requires hepatic metabolism to exert activity against gametocyte stages. OH-PQm exert modest antimalarial efficacy against parasite gametocytes; however, potency is enhanced ca.1000 fold in the presence of cytochrome P450 NADPH:oxidoreductase (CPR) from the liver and bone marrow. Enhancement of OH-PQm efficacy is due to the direct reduction of quinoneimine metabolites by CPR with the concomitant and excessive generation of H2O2, leading to parasite killing. This detailed understanding of the mechanism paves the way to rationally re-designed 8-aminoquinolines with improved pharmacological profiles.

Biology of Plasmodium falciparum gametocyte sex ratio and implications in malaria parasite transmission.

While significant advances have been made in understanding Plasmodium falciparum gametocyte biology and its relationship with malaria parasite transmission, the gametocyte sex ratio contribution to this process still remains a relevant research question. The present review discusses the biology of sex determination in P. falciparum, the underlying host and parasite factors, the sex specific susceptibility to drugs, the effect of sex ratio dynamics on malaria parasite transmission and the development of gametocyte sex specific diagnosis tools. Despite the inherent differences across several studies and approaches, the emerging picture highlights a potentially relevant contribution of the P. falciparum gametocyte sex ratio in the modulation of malaria parasite transmission. The increasing availability of molecular methods to measure gametocyte sex ratio will enable evaluation of important parameters, such as the impact of drug treatment on gametocyte sex ratio in vitro and in vivo as well as the changes of gametocyte sex ratios in natural infections, key steps towards elucidating how these parameters affect parasite infectiousness to the mosquito vectors.

The bacterial protein CNF1 as a new strategy against Plasmodium falciparum cytoadherence.

Plasmodium falciparum severe malaria causes more than 400,000 deaths every year. One feature of P. falciparum-parasitized erythrocytes (pRBC) leading to cerebral malaria (CM), the most dangerous form of severe malaria, is cytoadherence to endothelium and blockage of the brain microvasculature. Preventing ligand-receptor interactions involved in this process could inhibit pRBC sequestration and insurgence of severe disease whilst reversing existing cytoadherence could be a saving life adjunct therapy. Increasing evidence indicate the endothelial Rho signaling as a crucial player in malaria parasite cytoadherence. Therefore, we have used the cytotoxic necrotizing factor 1 (CNF1), an Escherichia coli protein able to modulate the activity of Cdc42, Rac, and Rho, three subfamilies of the Rho GTPases family, to study interactions between infected erythrocytes and cerebral endothelium in co-culture models. The main results are that CNF1 not only prevents cytoadherence but, more importantly, induces the detachment of pRBCs from endothelia monolayers. We first observed that CNF1 does affect neither parasite growth, nor the morphology and concentration of knobs that characterize the parasitized erythrocyte surface, as viewed by scanning electron microscopy. On the other hand, flow cytometry experiments show that cytoadherence reversion induced by CNF1 occurs in parallel with a decreased ICAM-1 receptor expression on the cell surface, suggesting the involvement of a toxin-promoted endocytic activity in such a response. Furthermore, since the endothelial barrier functionality is compromised by P. falciparum, we conducted a permeability assay on endothelial cells, revealing the CNF1 capacity to restore the brain endothelial barrier integrity. Then, using pull-down assays and inhibitory studies, we demonstrated, for the first time, that CNF1 is able not only to prevent but also to cause the parasite detachment by simultaneously activating Rho, Rac and Cdc42 in endothelial cells. All in all our findings indicate that CNF1 may represent a potential novel therapeutic strategy for preventing neurological complications of CM.

Gametocytes of the Malaria Parasite Plasmodium falciparum Interact With and Stimulate Bone Marrow Mesenchymal Cells to Secrete Angiogenetic Factors.

The gametocytes of Plasmodium falciparum, responsible for the transmission of this malaria parasite from humans to mosquitoes, accumulate and mature preferentially in the human bone marrow. In the 10 day long sexual development of P. falciparum, the immature gametocytes reach and localize in the extravascular compartment of this organ, in contact with several bone marrow stroma cell types, prior to traversing the endothelial lining and re-entering in circulation at maturity. To investigate the host parasite interplay underlying this still obscure process, we developed an in vitro tridimensional co-culture system in a Matrigel scaffold with P. falciparum gametocytes and self-assembling spheroids of human bone marrow mesenchymal cells (hBM-MSCs). Here we show that this co-culture system sustains the full maturation of the gametocytes and that the immature, but not the mature, gametocytes adhere to hBM-MSCs via trypsin-sensitive parasite ligands exposed on the erythrocyte surface. Analysis of a time course of gametocytogenesis in the co-culture system revealed that gametocyte maturation is accompanied by the parasite induced stimulation of hBM-MSCs to secrete a panel of 14 cytokines and growth factors, 13 of which have been described to play a role in angiogenesis. Functional in vitro assays on human bone marrow endothelial cells showed that supernatants from the gametocyte mesenchymal cell co-culture system enhance ability of endothelial cells to form vascular tubes. These results altogether suggest that the interplay between immature gametocytes and hBM-MSCs may induce functional and structural alterations in the endothelial lining of the human bone marrow hosting the P. falciparum transmission stages.

Probabilistic data integration identifies reliable gametocyte-specific proteins and transcripts in malaria parasites.

Plasmodium gametocytes are the sexual forms of the malaria parasite essential for transmission to mosquitoes. To better understand how gametocytes differ from asexual blood-stage parasites, we performed a systematic analysis of available 'omics data for P. falciparum and other Plasmodium species. 18 transcriptomic and proteomic data sets were evaluated for the presence of curated "gold standards" of 41 gametocyte-specific versus 46 non-gametocyte genes and integrated using Bayesian probabilities, resulting in gametocyte-specificity scores for all P. falciparum genes. To illustrate the utility of the gametocyte score, we explored newly predicted gametocyte-specific genes as potential biomarkers of gametocyte carriage and exposure. We analyzed the humoral immune response in field samples against 30 novel gametocyte-specific antigens and found five antigens to be differentially recognized by gametocyte carriers as compared to malaria-infected individuals without detectable gametocytes. We also validated the gametocyte-specificity of 15 identified gametocyte transcripts on culture material and samples from naturally infected individuals, resulting in eight transcripts that were >1000-fold higher expressed in gametocytes compared to asexual parasites and whose transcript abundance allowed gametocyte detection in naturally infected individuals. Our integrated genome-wide gametocyte-specificity scores provide a comprehensive resource to identify targets and monitor P. falciparum gametocytemia.

Detection of Plasmodium falciparum male and female gametocytes and determination of parasite sex ratio in human endemic populations by novel, cheap and robust RTqPCR assays.

BACKGROUND: The presence of Plasmodium falciparum gametocytes in peripheral blood is essential for human to mosquito parasite transmission. The detection of submicroscopic infections with gametocytes and the estimation of the gametocyte sex ratio are crucial to assess the human host potential ability to infect mosquitoes and transmit malaria parasites. AIM AND OBJECTIVES: The aim of this work was to develop sensitive and cheap Real Time qPCR assays for large-scale epidemiological surveys, based on detection and amplification of gametocyte sex specific transcripts selected from the literature: the female-specific pfs25 and pf glycerol kinase (pfGK) and the male-specific pfs230p and pf13 transcripts. METHODS: RTqPCR assays were used to test the gametocyte- and sex-specific expression of the target genes using asexual stages of the gametocyteless parasite clone F12 and FACS purified male and female gametocytes of the PfDynGFP/P47mCherry line. Assays were performed on 50 blood samples collected during an epidemiological survey in the Soumousso village, Burkina Faso, West-Africa, and amplification of the human housekeeping gene 18S rRNA was employed to normalize RNA sample variability. RESULTS: SYBR Green assays were developed that showed higher sensitivity compared to Taqman assays at a reduced cost. RTqPCR results confirmed that expression of pfs25 and pfs230p are female and male-specific, respectively, and introduced two novel markers, the female-specific pfGK and the male-specific pf13. A formula was derived to calculate the ratio of male to female gametocytes based on the ratio of male to female transcript copy number. Use of these assays in the field samples showed, as expected, a higher sensitivity of RTqPCR compared to microscopy. Importantly, similar values of gametocyte sex-ratio were obtained in the field samples based on the four different target combinations. CONCLUSION: Novel, sensitive, cheap and robust molecular assays were developed for the detection and quantification of female and male P. falciparum gametocytes. In particular, the RTqPCR assays based on the female-specific pfs25 and the newly described male gametocyte-specific pf13 transcripts, including normalization by the human 18S, reliably assess presence and abundance of female and male gametocytes and enable to determine their sex-ratio in human subjects in endemic areas.

A Molecular Assay to Quantify Male and Female Plasmodium falciparum Gametocytes: Results From 2 Randomized Controlled Trials Using Primaquine for Gametocyte Clearance.

Background: Single low-dose primaquine (PQ) reduces Plasmodium falciparum infectivity before it impacts gametocyte density. Here, we examined the effect of PQ on gametocyte sex ratio as a possible explanation for this early sterilizing effect. Methods: Quantitative reverse-transcription polymerase chain reaction assays were developed to quantify female gametocytes (targeting Pfs25 messenger RNA [mRNA]) and male gametocytes (targeting Pf3D7_1469900 mRNA) in 2 randomized trials in Kenya and Mali, comparing dihydroartemisinin-piperaquine (DP) alone to DP with PQ. Gametocyte sex ratio was examined in relation to time since treatment and infectivity to mosquitoes. Results: In Kenya, the median proportion of male gametocytes was 0.33 at baseline. Seven days after treatment, gametocyte density was significantly reduced in the DP-PQ arm relative to the DP arm (females: 0.05% [interquartile range {IQR}, 0.0-0.7%] of baseline; males: 3.4% [IQR, 0.4%-32.9%] of baseline; P < .001). Twenty-four hours after treatment, gametocyte sex ratio became male-biased and was not significantly different between the DP and DP-PQ groups. In Mali, there was no significant difference in sex ratio between the DP and DP-PQ groups (>0.125 mg/kg) 48 hours after treatment, and gametocyte sex ratio was not associated with mosquito infection rates. Conclusions: The early sterilizing effects of PQ may not be explained by the preferential clearance of male gametocytes and may be due to an effect on gametocyte fitness.

Ned-19 inhibition of parasite growth and multiplication suggests a role for NAADP mediated signalling in the asexual development of Plasmodium falciparum.

BACKGROUND: Although malaria is a preventable and curable human disease, millions of people risk to be infected by the Plasmodium parasites and to develop this illness. Therefore, there is an urgent need to identify new anti-malarial drugs. Ca2+ signalling regulates different processes in the life cycle of Plasmodium falciparum, representing a suitable target for the development of new drugs. RESULTS: This study investigated for the first time the effect of a highly specific inhibitor of nicotinic acid adenine dinucleotide phosphate (NAADP)-induced Ca2+ release (Ned-19) on P. falciparum, revealing the inhibitory effect of this compound on the blood stage development of this parasite. Ned-19 inhibits both the transition of the parasite from the early to the late trophozoite stage and the ability of the late trophozoite to develop to the multinucleated schizont stage. In addition, Ned-19 affects spontaneous intracellular Ca2+ oscillations in ring and trophozoite stage parasites, suggesting that the observed inhibitory effects may be associated to regulation of intracellular Ca2+ levels. CONCLUSIONS: This study highlights the inhibitory effect of Ned-19 on progression of the asexual life cycle of P. falciparum. The observation that Ned-19 inhibits spontaneous Ca2+ oscillations suggests a potential role of NAADP in regulating Ca2+ signalling of P. falciparum.

Hexahydroquinolines are antimalarial candidates with potent blood-stage and transmission-blocking activity.

Antimalarial compounds with dual therapeutic and transmission-blocking activity are desired as high-value partners for combination therapies. Here, we report the identification and characterization of hexahydroquinolines (HHQs) that show low nanomolar potency against both pathogenic and transmissible intra-erythrocytic forms of the malaria parasite Plasmodium falciparum. This activity translates into potent transmission-blocking potential, as shown by in vitro male gamete formation assays and reduced oocyst infection and prevalence in Anopheles mosquitoes. In vivo studies illustrated the ability of lead HHQs to suppress Plasmodium berghei blood-stage parasite proliferation. Resistance selection studies, confirmed by CRISPR-Cas9-based gene editing, identified the digestive vacuole membrane-spanning transporter PfMDR1 (P. falciparum multidrug resistance gene-1) as a determinant of parasite resistance to HHQs. Haemoglobin and haem fractionation assays suggest a mode of action that results in reduced haemozoin levels and might involve inhibition of host haemoglobin uptake into intra-erythrocytic parasites. Furthermore, parasites resistant to HHQs displayed increased susceptibility to several first-line antimalarial drugs, including lumefantrine, confirming that HHQs have a different mode of action to other antimalarials drugs for which PfMDR1 is known to confer resistance. This work evokes therapeutic strategies that combine opposing selective pressures on this parasite transporter as an approach to countering the emergence and transmission of multidrug-resistant P. falciparum malaria.

The emerging role of the human bone marrow as a privileged developmental niche for the transmission stages of the malaria parasite Plasmodium falciparum. Commentary.

The spread of malaria relies on the ability of the Plasmodium parasites to be transmitted from infected individuals to the Anopheles mosquito vectors. Recent work on the most lethal of the malaria parasites, Plasmodium falciparum, identified the infected human bone marrow as a preferential site for the localization and maturation of the parasite transmission stages, the gametocytes. These findings unveil a complex host parasite interplay and an unsuspected role of the bone marrow microenvironment in the successful transmission of the malaria parasite and have major implications in developing and targeting future interventions to block the transmission of P. falciparum.