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Summary Dormant liver stage forms (hypnozoites) of the malaria parasite Plasmodium vivax present major hurdles to control and eradicate infection. Despite major research efforts, the molecular composition of hypnozoites remains ill defined. Here, we applied a combination of state‐of‐the‐art technologies to generate the first transcriptome of hypnozoites. We developed a robust laser dissection microscopy protocol to isolate individual Plasmodium cynomolgi hypnozoites and schizonts from infected monkey hepatocytes and optimized RNA‐seq analysis to obtain the first transcriptomes of these stages. Comparative transcriptomic analysis identified 120 transcripts as being differentially expressed in the hypnozoite stage relative to the dividing liver schizont, with 69 and 51 mRNAs being up‐ or down‐regulated, respectively, in the hypnozoites. This lead to the identification of potential markers of commitment to and maintenance of the dormant state of the hypnozoite including three transcriptional regulators of the ApiAP2 family, one of which is unique to P. cynomolgi and P. vivax, and the global translational repressor, eIF2a kinase eIK2, all of which are upregulated in the hypnozoite. Together, this work not only provides a primary experimentally‐derived list of molecular markers of hypnozoites but also identifies transcriptional and posttranscriptional regulation of gene expression as potentially being key to establishing and maintaining quiescence. Quiescent liver stage forms of the malaria parasite Plasmodium vivax, which lead to a majority of malaria relapses, have thus far been refractory to molecular characterization. To overcome this, we developed a coupled immunostaining‐laser capture microdissection approach and performed transcriptomics (using RNA‐seq) of dividing and quiescent liver parasites (P. cynomolgi). Differential expression analysis identified potential, unique biomarkers of the quiescent state, which could aid both phenotypic characterization and drug design.

Plasmodium vivax infections often recur due to relapse of hypnozoites from the liver. In malaria-endemic areas, tools to distinguish relapse from reinfection are needed. We applied amplicon deep sequencing to P. vivax isolates from 78 Cambodian volunteers, nearly one-third of whom suffered recurrence at a median of 68 days. Deep sequencing at a highly variable region of the P. vivax merozoite surface protein 1 gene revealed impressive diversity—generating 67 unique haplotypes and detecting on average 3.6 cocirculating parasite clones within individuals, compared to 2.1 clones detected by a combination of 3 microsatellite markers. This diversity enabled a scheme to classify over half of recurrences as probable relapses based on the low probability of reinfection by multiple recurring variants. In areas of high P. vivax diversity, targeted deep sequencing can help detect genetic signatures of relapse, key to evaluating antivivax interventions and achieving a better understanding of relapsereinfection epidemiology.

Targeted protein degradation (TPD) is a novel strategy for developing therapeutics against pathogens. Prior to causing malaria, Plasmodium parasites replicate within hepatocytes as liver stages, surrounded by a parasitophorous vacuole membrane (PVM). We hypothesized that TPD can be employed to trigger host-driven degradation of essential liver stage PVM proteins and lead to parasite death. To explore this, we took advantage of the proteolysis-targeting-chimera HaloPROTAC3, a molecule that recruits the host von Hippel-Lindau (VHL) E3 ligase to the HaloTag (HT). Parasites expressing HT fused to the host cytosol-exposed domain of the PVM protein UIS4 (UIS4-HT) were generated in Plasmodium berghei and Plasmodium cynomolgi , but only P. berghei UIS4-HT enabled productive liver stage infection experiments in vitro. Although HaloPROTAC3 triggered the degradation of HT proteins in host cells, it had no impact on the survival of P. berghei UIS4-HT liver stages. Furthermore, HaloPROTAC3 bound to P. berghei UIS4-HT but did not recruit VHL or trigger ubiquitination of the PVM. Overall, although this study did not establish whether host-driven TPD can degrade Plasmodium PVM proteins, it highlights the challenges of developing TPD approaches against novel targets and offers insights for advancing this therapeutic strategy against pathogens.

Initiatives to eradicate malaria have a good impact on P. falciparum malaria worldwide. P. vivax , however, still presents significant difficulties. This is due to its unique biological traits, which, in comparison to P. falciparum , pose serious challenges for malaria elimination approaches. P. vivax's numerous distinctive characteristics and its ability to live for weeks to years in liver cells in its hypnozoite form, which may elude the human immune system and blood-stage therapy and offer protection during mosquito-free seasons. Many malaria patients are not fully treated because of contraindications to primaquine use in pregnant and nursing women and are still vulnerable to P. vivax relapses, although there are medications that could radical cure P. vivax . Additionally, due to CYP2D6's highly variable genetic polymorphism, the pharmacokinetics of primaquine may be impacted. Due to their inability to metabolize PQ, some CYP2D6 polymorphism alleles can cause patients to not respond to treatment. Tafenoquine offers a radical treatment in a single dose that overcomes the potentially serious problem of poor adherence to daily primaquine. Despite this benefit, hemolysis of the early erythrocytes continues in individuals with G6PD deficiency until all susceptible cells have been eliminated. Field techniques such as microscopy or rapid diagnostic tests (RDTs) miss the large number of submicroscopic and/or asymptomatic infections brought on by reticulocyte tropism and the low parasitemia levels that accompany it. Moreover, P. vivax gametocytes grow more quickly and are much more prevalent in the bloodstream . P. vivax populations also have a great deal of genetic variation throughout their genome, which ensures evolutionary fitness and boosts adaptation potential. Furthermore, P. vivax fully develops in the mosquito faster than P. falciparum . These characteristics contribute to parasite reservoirs in the human population and facilitate faster transmission. Overall, no genuine chance of eradication is predicted in the next few years unless new tools for lowering malaria transmission are developed (i.e., malaria elimination and eradication). The challenging characteristics of P. vivax that impede the elimination and eradication of malaria are thus discussed in this article.

Malaria is a vector-borne disease that exacts a grave toll in the Global South. The epidemiology of Plasmodium vivax, the most geographically expansive agent of human malaria, is characterised by the accrual of a reservoir of dormant parasites known as hypnozoites. Relapses, arising from hypnozoite activation events, comprise the majority of the blood-stage infection burden, with implications for the acquisition of immunity and the distribution of superinfection. Here, we construct a novel model for the transmission of P. vivax that concurrently accounts for the accrual of the hypnozoite reservoir, (blood-stage) superinfection and the acquisition of immunity. We begin by using an infinite-server queueing network model to characterise the within-host dynamics as a function of mosquito-to-human transmission intensity, extending our previous model to capture a discretised immunity level. To model transmission-blocking and antidisease immunity, we allow for geometric decay in the respective probabilities of successful human-to-mosquito transmission and symptomatic blood-stage infection as a function of this immunity level. Under a hybrid approximation—whereby probabilistic within-host distributions are cast as expected population-level proportions—we couple host and vector dynamics to recover a deterministic compartmental model in line with Ross-Macdonald theory. We then perform a steady-state analysis for this compartmental model, informed by the (analytic) distributions derived at the within-host level. To characterise transient dynamics, we derive a reduced system of integrodifferential equations, likewise informed by our within-host queueing network, allowing us to recover population-level distributions for various quantities of epidemiological interest. In capturing the interplay between hypnozoite accrual, superinfection and acquired immunity—and providing, to the best of our knowledge, the most complete population-level distributions for a range of epidemiological values—our model provides insights into important, but poorly understood, epidemiological features of P. vivax.

Plasmodium vivax hypnozoites persist in the liver, cause malaria relapse and represent a major challenge to malaria elimination. Our previous transcriptomic study provided a novel molecular framework to enhance our understanding of the hypnozoite biology (Voorberg-van der Wel A, et al., 2017). In this dataset, we identified and characterized the Liver-Specific Protein 2 (LISP2) protein as an early molecular marker of liver stage development. Immunofluorescence analysis of hepatocytes infected with relapsing malaria parasites, in vitro (P. cynomolgi) and in vivo (P. vivax), reveals that LISP2 expression discriminates between dormant hypnozoites and early developing parasites. We further demonstrate that prophylactic drugs selectively kill all LISP2-positive parasites, while LISP2-negative hypnozoites are only sensitive to anti-relapse drug tafenoquine. Our results provide novel biological insights in the initiation of liver stage schizogony and an early marker suitable for the development of drug discovery assays predictive of anti-relapse activity.

The resilience of Plasmodium vivax , the most widely-distributed malaria-causing parasite in humans, is attributed to its ability to produce dormant liver forms known as hypnozoites, which can activate weeks, months, or even years after an initial mosquito bite. The factors underlying hypnozoite formation and activation are poorly understood, as is the parasite’s influence on the host hepatocyte. Here, we shed light on transcriptome-wide signatures of both the parasite and the infected host cell by sequencing over 1,000 P. vivax -infected hepatocytes at single-cell resolution. We distinguish between replicating schizonts and hypnozoites at the transcriptional level, identifying key differences in transcripts encoding for RNA-binding proteins associated with cell fate. In infected hepatocytes, we show that genes associated with energy metabolism and antioxidant stress response are upregulated, and those involved in the host immune response downregulated, suggesting both schizonts and hypnozoites alter the host intracellular environment. The transcriptional markers in schizonts, hypnozoites, and infected hepatocytes revealed here pinpoint potential factors underlying dormancy and can inform therapeutic targets against P. vivax liver-stage infection.

One of the key obstacles to malaria elimination is largely attributed to Plasmodium vivax’s ability to form resilient hypnozoites in the host liver that cause relapsing infections. As a result, interruption of P. vivax transmission is difficult. P. vivax transmission occurs in Duffy-positive individuals and have been mainly thought to be absent in Africa. However, increasing studies using molecular tools detected P. vivax among Duffy-negative individuals in various African countries. Studies on the African P. vivax has been severely limited because most of malaria control program focus mainly on falciparum malaria. In addition, there is a scarcity of laboratory infrastructures to overcome the biological obstacles posed by P. vivax . Herein, we established field transmission of Ethiopian P. vivax for routine sporozoite supply followed by liver stage infection in Mali. Furthermore, we evaluated local P. vivax hypnozoites and schizonts susceptibilities to reference antimalarial drugs. The study enabled the assessment of local African P. vivax hypnozoite production dynamics. Our data displayed the ability of the African P. vivax to produce hypnozoite forms ex-vivo at different rates per field isolate. We report that while tafenoquine (1µM) potently inhibited both hypnozoites and schizont forms; atovaquone (0.25µM) and the phosphatidylinositol-4-OH kinase (PI4K)-specific inhibitor KDU691 (0.5µM) showed no activity against hypnozoites forms. Unlike hypnozoites forms, P. vivax schizont stages were fully susceptible to both atovaquone (0.25µM) and the (PI4K)-specific inhibitor KDU691 (0.5µM). Together, the data revealed the importance of the local platform for further biological investigation and implementation of drug discovery program on the African P. vivax clinical isolates.

Clinical trial and individual patient treatment outcomes have produced accumulating evidence that effective primaquine (PQ) treatment of Plasmodium vivax and P. ovale liver stage hypnozoites is associated with genetic variation in the human cytochrome P450 gene, CYP2D6 . Successful PQ treatment of individual and population-wide infections by the Plasmodium species that generate these dormant liver stage forms is likely to be necessary to reach elimination of malaria caused by these parasites globally. Optimizing safe and effective PQ treatment will require coordination of efforts between the malaria and pharmacogenomics research communities.

A curious aspect of the evolution of the hypnozoite theory of malarial relapse is its transmogrification from theory into ‘fact’, this being of historical, linguistic, scientific and sociological interest. As far as it goes, the hypnozoite explanation for relapse is almost certainly correct. I contend, however, that many of the genotypically homologous, non-reinfection, relapse-like Plasmodium vivax recurrences that researchers ascribe to hypnozoite activation are probably hypnozoite-independent. Indeed, some malariologists are starting to recognize that homologous P. vivax recurrences have most likely been overattributed to activation of hypnozoites. Hitherto identified, non-hypnozoite, possible plasmodial sources of recurrence that must be considered, besides circulating erythrocytic stages, include parasites in splenic dendritic cells, other cells in the spleen (in addition to infected erythrocytes there), bone marrow (importantly) and the skin. I argue that we need to take into account the possibility of a dual or multiple extra-vascular origin of P. vivax non-reinfection recurrences, not arbitrarily discount it. The existence of a P. vivax reservoir(s) is a topical subject and one of practical importance for malaria eradication. Pertinent drug-associated matters are also discussed, as is the dormancy-related significance of clues provided by blood-stage-induced malarial infection.