Comparison of molecular surveillance methods to assess changes in the population genetics of Plasmodium falciparum in high transmission.

A major motivation for developing molecular methods for malaria surveillance is to measure the impact of control interventions on the population genetics of Plasmodium falciparum as a potential marker of progress towards elimination. Here we assess three established methods (i) single nucleotide polymorphism (SNP) barcoding (panel of 24-biallelic loci), (ii) microsatellite genotyping (panel of 12-multiallelic loci), and (iii) varcoding (fingerprinting var gene diversity, akin to microhaplotyping) to identify changes in parasite population genetics in response to a short-term indoor residual spraying (IRS) intervention. Typical of high seasonal transmission in Africa, multiclonal infections were found in 82.3% (median 3; range 1-18) and 57.8% (median 2; range 1-12) of asymptomatic individuals pre- and post-IRS, respectively, in Bongo District, Ghana. Since directly phasing multilocus haplotypes for population genetic analysis is not possible for biallelic SNPs and microsatellites, we chose ~200 low-complexity infections biased to single and double clone infections for analysis. Each genotyping method presented a different pattern of change in diversity and population structure as a consequence of variability in usable data and the relative polymorphism of the molecular markers (i.e., SNPs < microsatellites < var). Varcoding and microsatellite genotyping showed the overall failure of the IRS intervention to significantly change the population structure from pre-IRS characteristics (i.e., many diverse genomes of low genetic similarity). The 24-SNP barcode provided limited information for analysis, largely due to the biallelic nature of SNPs leading to a high proportion of double-allele calls and a view of more isolate relatedness compared to microsatellites and varcoding. Relative performance, suitability, and cost-effectiveness of the methods relevant to sample size and local malaria elimination in high-transmission endemic areas are discussed.

Measuring changes in Plasmodium falciparum census population size in response to sequential malaria control interventions.

Here we introduce a new endpoint "census population size" to evaluate the epidemiology and control of Plasmodium falciparum infections, where the parasite, rather than the infected human host, is the unit of measurement. To calculate census population size, we rely on a definition of parasite variation known as multiplicity of infection (MOIvar), based on the hyper-diversity of the var multigene family. We present a Bayesian approach to estimate MOIvar from sequencing and counting the number of unique DBLα tags (or DBLα types) of var genes, and derive from it census population size by summation of MOIvar in the human population. We track changes in this parasite population size and structure through sequential malaria interventions by indoor residual spraying (IRS) and seasonal malaria chemoprevention (SMC) from 2012 to 2017 in an area of high-seasonal malaria transmission in northern Ghana. Following IRS, which reduced transmission intensity by > 90% and decreased parasite prevalence by ~40-50%, significant reductions in var diversity, MOIvar, and population size were observed in ~2,000 humans across all ages. These changes, consistent with the loss of diverse parasite genomes, were short lived and 32-months after IRS was discontinued and SMC was introduced, var diversity and population size rebounded in all age groups except for the younger children (1-5 years) targeted by SMC. Despite major perturbations from IRS and SMC interventions, the parasite population remained very large and retained the var population genetic characteristics of a high-transmission system (high var diversity; low var repertoire similarity) demonstrating the resilience of P. falciparum to short-term interventions in high-burden countries of sub-Saharan Africa.

Indoor residual spraying with a non-pyrethroid insecticide reduces the reservoir of Plasmodium falciparum in a high-transmission area in northern Ghana.

High-malaria burden countries in sub-Saharan Africa are shifting from malaria control towards elimination. Hence, there is need to gain a contemporary understanding of how indoor residual spraying (IRS) with non-pyrethroid insecticides when combined with long-lasting insecticidal nets (LLINs) impregnated with pyrethroid insecticides, contribute to the efforts of National Malaria Control Programmes to interrupt transmission and reduce the reservoir of Plasmodium falciparum infections across all ages. Using an interrupted time-series study design, four age-stratified malariometric surveys, each of ~2,000 participants, were undertaken pre- and post-IRS in Bongo District, Ghana. Following the application of three-rounds of IRS, P. falciparum transmission intensity declined, as measured by a >90% reduction in the monthly entomological inoculation rate. This decline was accompanied by reductions in parasitological parameters, with participants of all ages being significantly less likely to harbor P. falciparum infections at the end of the wet season post-IRS (aOR = 0.22 [95% CI: 0.19-0.26], p-value < 0.001). In addition, multiplicity of infection (MOI var ) was measured using a parasite fingerprinting tool, designed to capture within-host genome diversity. At the end of the wet season post-IRS, the prevalence of multi-genome infections declined from 75.6% to 54.1%. This study demonstrates that in areas characterized by high seasonal malaria transmission, IRS in combination with LLINs can significantly reduce the reservoir of P. falciparum infection. Nonetheless despite this success, 41.6% of the population, especially older children and adolescents, still harboured multi-genome infections. Given the persistence of this diverse reservoir across all ages, these data highlight the importance of sustaining vector control in combination with targeted chemotherapy to move high-transmission settings towards pre-elimination. This study also points to the benefits of molecular surveillance to ensure that incremental achievements are not lost and that the goals advocated for in the WHO's High Burden to High Impact strategy are realized.

The impact of indoor residual spraying on Plasmodium falciparum microsatellite variation in an area of high seasonal malaria transmission in Ghana, West Africa.

Here, we report the first population genetic study to examine the impact of indoor residual spraying (IRS) on Plasmodium falciparum in humans. This study was conducted in an area of high seasonal malaria transmission in Bongo District, Ghana. IRS was implemented during the dry season (November-May) in three consecutive years between 2013 and 2015 to reduce transmission and attempt to bottleneck the parasite population in humans towards lower diversity with greater linkage disequilibrium. The study was done against a background of widespread use of long-lasting insecticidal nets, typical for contemporary malaria control in West Africa. Microsatellite genotyping with 10 loci was used to construct 392 P. falciparum multilocus infection haplotypes collected from two age-stratified cross-sectional surveys at the end of the wet seasons pre- and post-IRS. Three-rounds of IRS, under operational conditions, led to a >90% reduction in transmission intensity and a 35.7% reduction in the P. falciparum prevalence (p < .001). Despite these declines, population genetic analysis of the infection haplotypes revealed no dramatic changes with only a slight, but significant increase in genetic diversity (He : pre-IRS = 0.79 vs. post-IRS = 0.81, p = .048). Reduced relatedness of the parasite population (p < .001) was observed post-IRS, probably due to decreased opportunities for outcrossing. Spatiotemporal genetic differentiation between the pre- and post-IRS surveys (D = 0.0329 [95% CI: 0.0209 - 0.0473], p = .034) was identified. These data provide a genetic explanation for the resilience of P. falciparum to short-term IRS programmes in high-transmission settings in sub-Saharan Africa.

Tip-enhanced Raman scattering (TERS) from hemozoin crystals within a sectioned erythrocyte.

Tip-enhanced Raman scattering (TERS) is a powerful technique to obtain molecular information on a nanometer scale, however, the technique has been limited to cell surfaces, viruses, and isolated molecules. Here we show that TERS can be used to probe hemozoin crystals at less than 20 nm spatial resolution in the digestive vacuole of a sectioned malaria parasite-infected cell. The TERS spectra clearly show characteristic bands of hemozoin that can be correlated to a precise position on the crystal by comparison with the corresponding atomic force microscopy (AFM) image. These are the first recorded AFM images of hemozoin crystals inside malaria-infected cells and clearly show the hemozoin crystals protruding from the embedding medium. TERS spectra recorded of these crystals show spectral features consistent with a five-coordinate high-spin ferric heme complex, which include the electron density marker band ν(4) at 1373 cm(-1) and other porphyrin skeletal and ring breathing modes at approximately 1636, 1557, 1412, 1314, 1123, and 1066 cm(-1). These results demonstrate the potential of the AFM/TERS technique to obtain nanoscale molecular information within a sectioned single cell. We foresee this approach paving the way to a new independent drug screening modality for detection of drugs binding to the hemozoin surface within the digestive vacuole of the malaria trophozoite.

Cryo transmission X-ray imaging of the malaria parasite, P. falciparum.

Cryo transmission X-ray microscopy in the "water window" of photon energies has recently been introduced as a method that exploits the natural contrast of biological samples. We have used cryo tomographic X-ray imaging of the intra-erythrocytic malaria parasite, Plasmodium falciparum, to undertake a survey of the cellular features of this important human pathogen. We examined whole hydrated cells at different stages of growth and defined some of the structures with different X-ray density, including the parasite nucleus, cytoplasm, digestive vacuole and the hemoglobin degradation product, hemozoin. As the parasite develops from an early cup-shaped morphology to a more rounded shape, puncta of hemozoin are formed; these coalesce in the mature trophozoite into a central compartment. In some trophozoite stage parasites we observed invaginations of the parasite surface and, using a selective permeabilization process, showed that these remain connected to the RBC cytoplasm. Some of these invaginations have large openings consistent with phagocytic structures and we observed independent endocytic vesicles in the parasite cytoplasm which appear to play a role in hemoglobin uptake. In schizont stage parasites staggered mitosis was observed and X-ray-dense lipid-rich structures were evident at their apical ends of the developing daughter cells. Treatment of parasites with the antimalarial drug artemisinin appears to affect parasite development and their ability to produce the hemoglobin breakdown product, hemozoin.

Hematin-hematin self-association states involved in the formation and reactivity of the malaria parasite pigment, hemozoin.

The malaria parasite pigment, hemozoin, is a crystal of ferriprotoporphyrin IX (FP-Fe(III)), a product of hemoglobin digestion. Hemozoin formation is essential for FP-Fe(III) detoxification in the parasite; it is the main target of quinoline antimalarials and can modulate immune and inflammation responses. To gain further insight into the likely mechanisms of crystal formation and hemozoin reactivity, we have reanalyzed the crystal structure data for beta-hematin and solved the crystal structure of Plasmodium falciparum hemozoin. The analysis reveals that the structures are very similar and highlights two previously unexplored modes of FP-Fe(III) self-association involving pi-pi interactions that may initiate crystal formation and help to stabilize the extended structure. Hemozoin can be considered to be a crystal composed of pi-pi dimers stabilized by iron-carboxylate linkages. As a result, it is predicted that two surfaces of the crystal would consist of pi-pi dimers with Fe(III) partly exposed to solvent and capable of undergoing redox reactions. Accordingly, we demonstrate that the crystal possesses both general peroxidase activity and the ability to cause lipid oxidation.

Whole cell imaging reveals novel modular features of the exomembrane system of the malaria parasite, Plasmodium falciparum.

During its intra-erythrocytic development Plasmodium falciparum establishes a membrane network beyond its own limiting membrane in the cytoplasm of its host. These membrane structures play an important role in the trafficking of virulence proteins to the erythrocyte surface, however their ultrastructure is only partly defined and there is on-going debate regarding their origin, organisation and connectivity. We have used two whole cell imaging modalities to explore the topography of parasitised erythrocytes. Three-dimensional structured illumination microscopy provides resolution beyond the optical diffraction limit and permits analysis of fluorescently labelled whole cells. Immunoelectron tomography offers the possibility of high resolution imaging of individual ultrastructural features in a cellular context. Combined with serial sectioning and immunogold labelling, this technique permits precise mapping of whole cell architecture. We show that the P. falciparum exported secretory system comprises a series of modular units, comprising flattened cisternae, known as Maurer's clefts, tubular connecting elements, two different vesicle populations and electron-dense structures that have fused with the erythrocyte membrane. The membrane network is not continuous, pointing to an important role for vesicle-mediated transport in the delivery of cargo to different destinations in the host cell.

Resonance Raman microscopy in combination with partial dark-field microscopy lights up a new path in malaria diagnostics.

Our goal is to produce a rapid and accurate diagnostic tool for malaria using resonance Raman spectroscopy to detect small inclusions of haemozoin in Plasmodium falciparum infected red blood cells. In pursuit of this aim we serendipitously discovered a partial dark-field effect generated by our experimental setup, which helps identify in thick blood films potential parasites that are normally difficult to see with conventional bright-field microscopy. The haemozoin deposits 'light up' and these can be selectively targeted with the Raman microscope to confirm the presence or absence of haemozoin by the strong 1569 cm(-1) band, which is a marker for haemozoin. With newly developed imaging Raman microscopes incorporating ultra-sensitive rapid readout CCDs it is possible to obtain spectra with a good signal-to-noise ratio in 1 second. Moreover, images from a smear of potentially infected cells can be recorded and analysed with multivariate methods. The reconstructed images show what appear to be sub-micron-inclusions of haemozoin in some cells indicating that the technique has potential to identify low pigmented forms of the parasite including early trophozoite-stage infected cells. Further work is required to unambiguously confirm the presence of such forms through systematic staining but the results are indeed promising and may lead to the development of a new Raman-based malaria diagnostic.

Discriminating the intraerythrocytic lifecycle stages of the malaria parasite using synchrotron FT-IR microspectroscopy and an artificial neural network.

Synchrotron Fourier transform infrared (FT-IR) spectra of fixed single erythrocytes infected with Plasmodium falciparum at different stages of the intraerythrocytic cycle are presented for the first time. Bands assigned to the hemozoin moiety at 1712, 1664, and 1209 cm(-1) are observed in FT-IR difference spectra between uninfected erythrocytes and infected trophozoites. These bands are also found to be important contributors in separating the trophozoite spectra from the uninfected cell spectra in principal components analysis. All stages of the intraerythrocytic lifecycle of the malarial parasite, including the ring and schizont stage, can be differentiated by visual inspection of the C-H stretching region (3100-2800 cm(-1)) and by using principal components analysis. Bands at 2922, 2852, and 1738 cm(-1) assigned to the nu(asym)(CH(2) acyl chain lipids), nu(sym)(CH(2) acyl chain lipids), and the ester carbonyl band, respectively, increase as the parasite matures from its early ring stage to the trophozoite and finally to the schizont stage. Training of an artificial neural network showed that excellent automated spectroscopic discrimination between P. falciparum-infected cells and the control cells is possible. FT-IR difference spectra indicate a change in the production of unsaturated fatty acids as the parasite matures. The ring stage spectrum shows bands associated with cis unsaturated fatty acids. The schizont stage spectrum displays no evidence of cis bands and suggests an increase in saturated fatty acids. These results demonstrate that different phases of the P. falciparum intraerthyrocytic life cycle are characterized by different lipid compositions giving rise to distinct spectral profiles in the C-H stretching region. This insight paves the way for an automated infrared-based technology capable of diagnosing malaria at all intraerythrocytic stages of the parasite's life cycle.

Resonance Raman spectroscopy can detect structural changes in haemozoin (malaria pigment) following incubation with chloroquine in infected erythrocytes.

Resonance Raman spectroscopy was applied to monitor the effects of chloroquine (CQ) treatment on cultures of Plasmodium falciparum trophozoites. A number of bands assigned to A(1g) and B(1g) modes characteristic of the haemozoin aggregate are reduced in intensity in the CQ-treated cells, however, no bands from the CQ are observed. The intensity changes are attributed to intermolecular drug binding of the CQ in a sandwich type complex between ferriprotoporphyrin IX (FePPIX) dimer units. It is postulated that the CQ binds via pi-pi interactions between adjacent and orientated porphyrins thereby disrupting the haemozoin aggregate and reducing excitonic interactions between adjacent haems. The results show the potential of Raman microscopy as a screening tool for FePPIX:drug interactions in live cells.

Electron tomography of the Maurer's cleft organelles of Plasmodium falciparum-infected erythrocytes reveals novel structural features.

During intraerythrocytic development, the human malaria parasite, Plasmodium falciparum, establishes membrane-bound compartments, known as Maurer's clefts, outside the confines of its own plasma membrane. The Maurer's compartments are thought to be a crucial component of the machinery for protein sorting and trafficking; however, their ultrastructure is only partly defined. We have used electron tomography to image Maurer's clefts of 3D7 strain parasites. The compartments are revealed as flattened structures with a translucent lumen and a more electron-dense coat. They display a complex and convoluted morphology, and some regions are modified with surface nodules, each with a circular cross-section of approximately 25 nm. Individual 25 nm vesicle-like structures are also seen in the erythrocyte cytoplasm and associated with the red blood cell membrane. The Maurer's clefts are connected to the red blood cell membrane by regions with extended stalk-like profiles. Immunogold labelling with specific antibodies confirms differential labelling of the Maurer's clefts and the parasitophorous vacuole and erythrocyte membranes. Spot fluorescence photobleaching was used to demonstrate the absence of a lipid continuum between the Maurer's clefts and parasite membranes and the host plasma membrane.

Primaquine synergises the activity of chloroquine against chloroquine-resistant P. falciparum.

In recent years, resistance to the antimalarial drug, chloroquine, has become widespread. It is, therefore, imperative to find compounds that could replace chloroquine or work synergistically with this drug to overcome chloroquine resistance. We have examined the interaction between chloroquine, a 4-aminoquinoline, and a number of 8-aminoquinolines, including primaquine, a drug that is widely used to treat Plasmodium vivax infections. We find that primaquine is a potent synergiser of the activity of chloroquine against chloroquine-resistant Plasmodium falciparum. Analysis of matched transfectants expressing mutant and wild-type alleles of the P. falciparum chloroquine resistance transporter (PfCRT) indicate that primaquine exerts its activity by blocking PfCRT, and thus enhancing chloroquine accumulation. Our data suggest that a novel formulation of two antimalarial drugs already licensed for use in humans could be used to treat chloroquine-resistant parasites.