Understanding Braess' Paradox in power grids.

The ongoing energy transition requires power grid extensions to connect renewable generators to consumers and to transfer power among distant areas. The process of grid extension requires a large investment of resources and is supposed to make grid operation more robust. Yet, counter-intuitively, increasing the capacity of existing lines or adding new lines may also reduce the overall system performance and even promote blackouts due to Braess' paradox. Braess' paradox was theoretically modeled but not yet proven in realistically scaled power grids. Here, we present an experimental setup demonstrating Braess' paradox in an AC power grid and show how it constrains ongoing large-scale grid extension projects. We present a topological theory that reveals the key mechanism and predicts Braessian grid extensions from the network structure. These results offer a theoretical method to understand and practical guidelines in support of preventing unsuitable infrastructures and the systemic planning of grid extensions.

CRISPR/Cas9-engineered inducible gametocyte producer lines as a valuable tool for Plasmodium falciparum malaria transmission research.

The malaria parasite Plasmodium falciparum replicates inside erythrocytes in the blood of infected humans. During each replication cycle, a small proportion of parasites commits to sexual development and differentiates into gametocytes, which are essential for parasite transmission via the mosquito vector. Detailed molecular investigation of gametocyte biology and transmission has been hampered by difficulties in generating large numbers of these highly specialised cells. Here, we engineer P. falciparum NF54 inducible gametocyte producer (iGP) lines for the routine mass production of synchronous gametocytes via conditional overexpression of the sexual commitment factor GDV1. NF54/iGP lines consistently achieve sexual commitment rates of 75% and produce viable gametocytes that are transmissible by mosquitoes. We also demonstrate that further genetic engineering of NF54/iGP parasites is a valuable tool for the targeted exploration of gametocyte biology. In summary, we believe the iGP approach developed here will greatly expedite basic and applied malaria transmission stage research.

The catalytic subunit of Plasmodium falciparum casein kinase 2 is essential for gametocytogenesis.

Casein kinase 2 (CK2) is a pleiotropic kinase phosphorylating substrates in different cellular compartments in eukaryotes. In the malaria parasite Plasmodium falciparum, PfCK2 is vital for asexual proliferation of blood-stage parasites. Here, we applied CRISPR/Cas9-based gene editing to investigate the function of the PfCK2α catalytic subunit in gametocytes, the sexual forms of the parasite that are essential for malaria transmission. We show that PfCK2α localizes to the nucleus and cytoplasm in asexual and sexual parasites alike. Conditional knockdown of PfCK2α expression prevented the transition of stage IV into transmission-competent stage V gametocytes, whereas the conditional knockout of pfck2a completely blocked gametocyte maturation already at an earlier stage of sexual differentiation. In summary, our results demonstrate that PfCK2α is not only essential for asexual but also sexual development of P. falciparum blood-stage parasites and encourage studies exploring PfCK2α as a potential target for dual-active antimalarial drugs.

PCR correction strategies for malaria drug trials: updates and clarifications.

Malaria drug trials conducted in endemic areas face a major challenge in their analysis because it is difficult to establish whether parasitaemia in blood samples collected after treatment indicate drug failure or a new infection acquired after treatment. It is therefore vital to reliably distinguish drug failures from new infections in order to obtain accurate estimates of drug failure rates. This distinction can be achieved for Plasmodium falciparum by comparing parasite genotypes obtained at the time of treatment (the baseline) and on the day of recurring parasitaemia. Such PCR correction is required to obtain accurate failure rates, even for new effective drugs. Despite the routine use of PCR correction in surveillance of drug resistance and in clinical drug trials, limitations inherent to the molecular genotyping methods have led some researchers to question the validity of current PCR correction strategies. Here we describe and discuss recent developments in these genotyping approaches, with a particular focus on method validation and limitations of the genotyping strategies. Our aim is to update scientists from public and private bodies who are working on the development, deployment, and surveillance of new malaria drugs. We aim to promote discussion around these issues and argue for the adoption of improved standardised PCR correction methodologies.

Multiplicity of Asymptomatic Plasmodium falciparum Infections and Risk of Clinical Malaria: A Systematic Review and Pooled Analysis of Individual Participant Data.

BACKGROUND: The malaria parasite Plasmodium falciparum holds an extensive genetic polymorphism. In this pooled analysis, we investigate how the multiplicity in asymptomatic P. falciparum infections-that is, the number of coinfecting clones-affects the subsequent risk of clinical malaria in populations living under different levels of transmission. METHODS: A systematic search of the literature was performed to identify studies in which P. falciparum infections were genotyped in asymptomatic individuals who were followed up prospectively regarding the incidence of clinical malaria. Individual participant data were pooled from 15 studies (n = 3736 individuals). RESULTS: Multiclonal asymptomatic infections were associated with a somewhat increased subsequent risk of clinical malaria in the youngest children, followed by an initial declining risk with age irrespective of transmission intensity. At approximately 5 years of age, the risk continued the gradual decline with age in high-transmission settings. However, in older children in moderate-, low-, and seasonal-transmission settings, multiclonal infections were either not significantly associated with the risk of subsequent febrile malaria or were associated with an increased risk. CONCLUSIONS: The number of clones in asymptomatic P. falciparum infections is associated with different risks of subsequent clinical malaria depending on age and transmission intensity.

The PHIST protein GEXP02 targets the host cytoskeleton during sexual development of Plasmodium falciparum.

A hallmark of the biology of Plasmodium falciparum blood stage parasites is their extensive host cell remodelling, facilitated by parasite proteins that are exported into the erythrocyte. Although this area has received extensive attention, only a few exported parasite proteins have been analysed in detail, and much of this remodelling process remains unknown, particularly for gametocyte development. Recent advances to induce high rates of sexual commitment enable the production of large numbers of gametocytes. We used this approach to study the Plasmodium helical interspersed subtelomeric (PHIST) protein GEXP02, which is expressed during sexual development. We show by immunofluorescence that GEXP02 is exported to the gametocyte-infected host cell periphery. Co-immunoprecipitation revealed potential interactions between GEXP02 and components of the erythrocyte cytoskeleton as well as other exported parasite proteins. This indicates that GEXP02 targets the erythrocyte cytoskeleton and is likely involved in its remodelling. GEXP02 knock-out parasites show no obvious phenotype during gametocyte maturation, transmission through mosquitoes, and hepatocyte infection, suggesting auxiliary or redundant functions for this protein. In summary, we performed a detailed cellular and biochemical analysis of a sexual stage-specific exported parasite protein using a novel experimental approach that is broadly applicable to study the biology of P. falciparum gametocytes.

Amplicon deep sequencing improves Plasmodium falciparum genotyping in clinical trials of antimalarial drugs.

Clinical trials monitoring malaria drug resistance require genotyping of recurrent Plasmodium falciparum parasites to distinguish between treatment failure and new infection occurring during the trial follow up period. Because trial participants usually harbour multi-clonal P. falciparum infections, deep amplicon sequencing (AmpSeq) was employed to improve sensitivity and reliability of minority clone detection. Paired samples from 32 drug trial participants were Illumina deep-sequenced for five molecular markers. Reads were analysed by custom-made software HaplotypR and trial outcomes compared to results from the previous standard genotyping method based on length-polymorphic markers. Diversity of AmpSeq markers in pre-treatment samples was comparable or higher than length-polymorphic markers. AmpSeq was highly reproducible with consistent quantification of co-infecting parasite clones within a host. Outcomes of the three best-performing markers, cpmp, cpp and ama1-D3, agreed in 26/32 (81%) of patients. Discordance between the three markers performed per sample was much lower by AmpSeq (six patients) compared to length-polymorphic markers (eleven patients). Using AmpSeq for discrimination of recrudescence and new infection in antimalarial drug trials provides highly reproducible and robust characterization of clone dynamics during trial follow-up. AmpSeq overcomes limitations inherent to length-polymorphic markers. Regulatory clinical trials of antimalarial drugs will greatly benefit from this unbiased typing method.

The Plasmodium falciparum Hsp70-x chaperone assists the heat stress response of the malaria parasite.

Plasmodium falciparum is the most lethal of human-infective malaria parasites. A hallmark of P. falciparum malaria is extensive remodeling of host erythrocytes by the parasite, which facilitates the development of virulence properties such as host cell adhesion to the endothelial lining of the microvasculature. Host remodeling is mediated by a large complement of parasite proteins exported to the erythrocyte; among them is a single heat shock protein (Hsp)70-class protein chaperone, P. falciparum Hsp70-x (PfHsp70-x). PfHsp70-x was previously shown to assist the development of virulent cytoadherence characteristics. Here, we show that PfHsp70-x also supports parasite growth under elevated temperature conditions that simulate febrile episodes, especially at the beginning of the parasite life cycle when most of host cell remodeling takes place. Biochemical and biophysical analyses of PfHsp70-x, including crystallographic structures of its catalytic domain and the J-domain of its stimulatory Hsp40 cochaperone, suggest that PfHsp70-x is highly similar to human Hsp70 chaperones endogenous to the erythrocyte. Nevertheless, our results indicate that selective inhibition of PfHsp70-x function using small molecules may be possible and highlight specific sites of its catalytic domain as potentially of high interest. We discuss the likely roles of PfHsp70-x and human chaperones in P. falciparum biology and how specific inhibitors may assist us in disentangling their relative contributions.-Day, J., Passecker, A., Beck, H.-P., Vakonakis, I. The Plasmodium falciparum Hsp70-x chaperone assists the heat stress response of the malaria parasite.

Host Cytoskeleton Remodeling throughout the Blood Stages of Plasmodium falciparum.

The asexual intraerythrocytic development of Plasmodium falciparum, causing the most severe form of human malaria, is marked by extensive host cell remodeling. Throughout the processes of invasion, intracellular development, and egress, the erythrocyte membrane skeleton is remodeled by the parasite as required for each specific developmental stage. The remodeling is facilitated by a plethora of exported parasite proteins, and the erythrocyte membrane skeleton is the interface of most of the observed interactions between the parasite and host cell proteins. Host cell remodeling has been extensively described and there is a vast body of information on protein export or the description of parasite-induced structures such as Maurer's clefts or knobs on the host cell surface. Here we specifically review the molecular level of each host cell-remodeling step at each stage of the intraerythrocytic development of P. falciparum We describe key events, such as invasion, knob formation, and egress, and identify the interactions between exported parasite proteins and the host cell cytoskeleton. We discuss each remodeling step with respect to time and specific requirement of the developing parasite to explain host cell remodeling in a stage-specific manner. Thus, we highlight the interaction with the host membrane skeleton as a key event in parasite survival.

Hemozoin-catalyzed precipitation polymerization as an assay for malaria diagnosis.

Methods to diagnose malaria are of paramount interest to eradicate the disease. Current methods have severe limitations, as they are either costly or not sensitive enough to detect low levels of parasitemia. Here we report an ultrasensitive, yet low-resource chemical assay for the detection and quantification of hemozoin, a biomarker of all Plasmodium species. Solubilized hemozoin catalyzes the atom transfer radical polymerization of N-isopropylacrylamide above the lower critical solution temperature of poly(N-isopropylacrylamide). The solution becomes turbid, which can be observed by naked eye and quantified by UV-visible spectroscopy. The rate of turbidity increase is proportional to the concentration of hemozoin, with a detection limit of 0.85 ng mL-1. Malaria parasites in human blood can be detected down to 10 infected red blood cells µL-1. The assay could potentially be applied as a point-of-care test. The signal-amplification of an analyte by biocatalytic precipitation polymerization represents a powerful approach in biosensing.

Immuno-gold silver staining assays on capillary-driven microfluidics for the detection of malaria antigens.

Accurate and affordable rapid diagnostic tests (RDTs) are indispensable but often lacking for many infectious diseases. Specifically, there is a lack of highly sensitive malaria RDTs that can detect low antigen concentration at the onset of infection. Here, we present a strategy to improve the sensitivity of malaria RDTs by using capillary-driven microfluidic chips and combining sandwich immunoassays with electroless silver staining. We used 5 µm fluorescent beads functionalized with capture antibodies (cAbs). These beads are self-assembled by capillary action in recessed "bead lanes", which cross the main flow path of chips microfabricated in Si and SU-8. The binding of analytes to detection antibodies (dAbs) and secondary antibodies (2ndAbs) conjugated to gold nanoparticles (NPs) allows the formation of a silver film on the beads. Such silver film masks the fluorescent core of the bead inversely proportional to the concentration of antigen in a sample. We illustrate this method using the recombinant malaria antigen Plasmodium falciparum histidine-rich-protein 2 (rPfHRP2) spiked in human serum. This antigen was a recombinant HRP2 protein expressed in Escherichia coli, which is also the standard reference material. The limit of detection (LOD) of our immunoassay was found to be less than 6 ng mL-1 of rPfHRP2 within 20 min, which is approaching the desired sensitivity needed in the Target Product Profile (TPP) for malaria elimination settings. The concept presented here is flexible and may also be utilized for implementing fluorescence immunoassays for the parallel detection of biomarkers on capillary-driven microfluidic chips.

Single-Dose Tafenoquine to Prevent Relapse of Plasmodium vivax Malaria.

BACKGROUND: Treatment of Plasmodium vivax malaria requires the clearing of asexual parasites, but relapse can be prevented only if dormant hypnozoites are cleared from the liver (a treatment termed "radical cure"). Tafenoquine is a single-dose 8-aminoquinoline that has recently been registered for the radical cure of P. vivax. METHODS: This multicenter, double-blind, double-dummy, parallel group, randomized, placebo-controlled trial was conducted in Ethiopia, Peru, Brazil, Cambodia, Thailand, and the Philippines. We enrolled 522 patients with microscopically confirmed P. vivax infection (>100 to <100,000 parasites per microliter) and normal glucose-6-phosphate dehydrogenase (G6PD) activity (with normal activity defined as ≥70% of the median value determined at each trial site among 36 healthy male volunteers who were otherwise not involved in the trial). All patients received a 3-day course of chloroquine (total dose of 1500 mg). In addition, patients were assigned to receive a single 300-mg dose of tafenoquine on day 1 or 2 (260 patients), placebo (133 patients), or a 15-mg dose of primaquine once daily for 14 days (129 patients). The primary outcome was the Kaplan-Meier estimated percentage of patients who were free from recurrence at 6 months, defined as P. vivax clearance without recurrent parasitemia. RESULTS: In the intention-to-treat population, the percentage of patients who were free from recurrence at 6 months was 62.4% in the tafenoquine group (95% confidence interval [CI], 54.9 to 69.0), 27.7% in the placebo group (95% CI, 19.6 to 36.6), and 69.6% in the primaquine group (95% CI, 60.2 to 77.1). The hazard ratio for the risk of recurrence was 0.30 (95% CI, 0.22 to 0.40) with tafenoquine as compared with placebo (P<0.001) and 0.26 (95% CI, 0.18 to 0.39) with primaquine as compared with placebo (P<0.001). Tafenoquine was associated with asymptomatic declines in hemoglobin levels, which resolved without intervention. CONCLUSIONS: Single-dose tafenoquine resulted in a significantly lower risk of P. vivax recurrence than placebo in patients with phenotypically normal G6PD activity. (Funded by GlaxoSmithKline and Medicines for Malaria Venture; DETECTIVE ClinicalTrials.gov number, NCT01376167 .).

Molecular assays for antimalarial drug resistance surveillance: A target product profile.

Antimalarial drug resistance is a major constraint for malaria control and elimination efforts. Artemisinin-based combination therapy is now the mainstay for malaria treatment. However, delayed parasite clearance following treatment with artemisinin derivatives has now spread in the Greater Mekong Sub region and may emerge or spread to other malaria endemic regions. This spread is of great concern for malaria control programmes, as no alternatives to artemisinin-based combination therapies are expected to be available in the near future. There is a need to strengthen surveillance systems for early detection and response to the antimalarial drug resistance threat. Current surveillance is mainly done through therapeutic efficacy studies; however these studies are complex and both time- and resource-intensive. For multiple common antimalarials, parasite drug resistance has been correlated with specific genetic mutations, and the molecular markers associated with antimalarial drug resistance offer a simple and powerful tool to monitor the emergence and spread of resistant parasites. Different techniques to analyse molecular markers associated with antimalarial drug resistance are available, each with advantages and disadvantages. However, procedures are not adequately harmonized to facilitate comparisons between sites. Here we describe the target product profiles for tests to analyse molecular markers associated with antimalarial drug resistance, discuss how use of current techniques can be standardised, and identify the requirements for an ideal product that would allow malaria endemic countries to provide useful spatial and temporal information on the spread of resistance.

Malaria and the 'last' parasite: how can technology help?

Malaria, together with HIV/AIDS, tuberculosis and hepatitis are the four most deadly infectious diseases globally. Progress in eliminating malaria has saved millions of lives, but also creates new challenges in detecting the 'last parasite'. Effective and accurate detection of malaria infections, both in symptomatic and asymptomatic individuals are needed. In this review, the current progress in developing new diagnostic tools to fight malaria is presented. An ideal rapid test for malaria elimination is envisioned with examples to demonstrate how innovative technologies can assist the global defeat against this disease. Diagnostic gaps where technology can bring an impact to the elimination campaign for malaria are identified. Finally, how a combination of microfluidic-based technologies and smartphone-based read-outs could potentially represent the next generation of rapid diagnostic tests is discussed.

A bead-based immunogold-silver staining assay on capillary-driven microfluidics.

Point-of-care (POC) diagnostics are critically needed for the detection of infectious diseases, particularly in remote settings where accurate and appropriate diagnosis can save lives. However, it is difficult to implement immunoassays, and specifically immunoassays relying on signal amplification using silver staining, into POC diagnostic devices. Effective immobilization of antibodies in such devices is another challenge. Here, we present strategies for immobilizing capture antibodies (cAbs) in capillary-driven microfluidic chips and implementing a gold-catalyzed silver staining reaction. We illustrate these strategies using a species/anti-species immunoassay and the capillary assembly of fluorescent microbeads functionalized with cAbs in "bead lanes", which are engraved in microfluidic chips. The microfluidic chips are fabricated in silicon (Si) and sealed with a dry film resist. Rabbit IgG antibodies in samples are captured on the beads and bound by detection antibodies (dAbs) conjugated to gold nanoparticles. The gold nanoparticles catalyze the formation of a metallic film of silver, which attenuates fluorescence from the beads in an analyte-concentration dependent manner. The performance of these immunoassays was found comparable to that of assays performed in 96 well microtiter plates using "classical" enzyme-linked immunosorbent assay (ELISA). The proof-of-concept method developed here can detect 24.6 ng mL-1 of rabbit IgG antibodies in PBS within 20 min, in comparison to 17.1 ng mL-1 of the same antibodies using a ~140-min-long ELISA protocol. Furthermore, the concept presented here is flexible and necessitate volumes of samples and reagents in the range of just a few microliters.

Challenges in Malaria Management and a Glimpse at Some Nanotechnological Approaches.

Malaria is a devastating infectious disease transmitted by mosquitoes, affecting millions of people and killing about half a million children each year. Despite tremendous progress in the control and elimination of malaria within the past years, there are still considerable challenges to be solved. To name a few, drug-resistant parasites, insecticide-resistant mosquitoes and the difficulty to formulate a potent malaria vaccine need to be addressed with new strategies to achieve the final goal of malaria eradication. Nanotechnology-researching and designing innovative structures at the nanoscale-is a promising contemporary technology that is being applied to a vast number of biomedical problems. In the case of malaria, nanotechnology provides tools to design strategies to target drug molecules to specific stages of the parasite, treat drug-resistant parasites, resolve severe malaria, increase vaccine efficacies and combinations thereof. This chapter introduces malaria, discusses current challenges of malaria control and relates these challenges to some potential solutions provided by the nanotechnology field.

Critical Evaluation of Molecular Monitoring in Malaria Drug Efficacy Trials and Pitfalls of Length-Polymorphic Markers.

Estimation of drug efficacy in antimalarial drug trials requires parasite genotyping to distinguish new infections from treatment failures. When using length-polymorphic molecular markers, preferential amplification of short fragments can compromise detection of coinfections, potentially leading to misclassification of treatment outcome. We quantified minority clone detectability and competition among msp1, msp2, and glurp amplicons using mixtures of Plasmodium falciparum strains and investigated the impact of template competition on genotyping outcomes in 44 paired field samples. Substantial amplification bias was detected for all three markers, with shorter fragments outperforming larger fragments. The strongest template competition was observed for the marker glurp Detection of glurp fragments in multiclonal infections was severely compromised. Eight of 44 sample pairs were identified as new infections by all three markers. Ten pairs were defined as new infections based on one marker alone, seven of which were defined by the questionable marker glurp The impact of size-dependent template competition on genotyping outcomes therefore calls for necessary amendments to the current WHO recommendations for PCR correction of malaria drug trial endpoints. Accuracy of genotyping outcomes could be improved by separate amplification reactions per allelic family and basing results on markers msp1 and msp2 first, with glurp only used to resolve discordant results.

Mycobacterium tuberculosis lineage 4 comprises globally distributed and geographically restricted sublineages.

Generalist and specialist species differ in the breadth of their ecological niches. Little is known about the niche width of obligate human pathogens. Here we analyzed a global collection of Mycobacterium tuberculosis lineage 4 clinical isolates, the most geographically widespread cause of human tuberculosis. We show that lineage 4 comprises globally distributed and geographically restricted sublineages, suggesting a distinction between generalists and specialists. Population genomic analyses showed that, whereas the majority of human T cell epitopes were conserved in all sublineages, the proportion of variable epitopes was higher in generalists. Our data further support a European origin for the most common generalist sublineage. Hence, the global success of lineage 4 reflects distinct strategies adopted by different sublineages and the influence of human migration.

Plasmodium Helical Interspersed Subtelomeric (PHIST) Proteins, at the Center of Host Cell Remodeling.

During the asexual cycle, Plasmodium falciparum extensively remodels the human erythrocyte to make it a suitable host cell. A large number of exported proteins facilitate this remodeling process, which causes erythrocytes to become more rigid, cytoadherent, and permeable for nutrients and metabolic products. Among the exported proteins, a family of 89 proteins, called the Plasmodium helical interspersed subtelomeric (PHIST) protein family, has been identified. While also found in other Plasmodium species, the PHIST family is greatly expanded in P. falciparum. Although a decade has passed since their first description, to date, most PHIST proteins remain uncharacterized and are of unknown function and localization within the host cell, and there are few data on their interactions with other host or parasite proteins. However, over the past few years, PHIST proteins have been mentioned in the literature at an increasing rate owing to their presence at various localizations within the infected erythrocyte. Expression of PHIST proteins has been implicated in molecular and cellular processes such as the surface display of PfEMP1, gametocytogenesis, changes in cell rigidity, and also cerebral and pregnancy-associated malaria. Thus, we conclude that PHIST proteins are central to host cell remodeling, but despite their obvious importance in pathology, PHIST proteins seem to be understudied. Here we review current knowledge, shed light on the definition of PHIST proteins, and discuss these proteins with respect to their localization and probable function. We take into consideration interaction studies, microarray analyses, or data from blood samples from naturally infected patients to combine all available information on this protein family.