Malaria relapses, resulting from the activation of quiescent hepatic hypnozoites of Plasmodium vivax and Plasmodium ovale, hinder global efforts to control and eliminate malaria. As primaquine, the only drug capable of eliminating hypnozoites, is unsuitable for mass administration, an alternative drug is needed urgently. Currently, analyses of hypnozoites, including screening of compounds that would eliminate them, can only be made using common macaque models, principally Macaca rhesus and Macaca fascicularis, experimentally infected with the relapsing Plasmodium cynomolgi. Here, we present a protocol for long-term in vitro cultivation of P. cynomolgi-infected M. fascicularis primary hepatocytes during which hypnozoites persist and activate to resume normal development. In a proof-of-concept experiment, we obtained evidence that exposure to an inhibitor of histone modification enzymes implicated in epigenetic control of gene expression induces an accelerated rate of hypnozoite activation. The protocol presented may further enable investigations of hypnozoite biology and the search for compounds that kill hypnozoites or disrupt their quiescence.

BackgroundAmongst the Plasmodium species in humans, only P. vivax and P. ovale produce latent hepatic stages called hypnozoites, which are responsible for malaria episodes long after a mosquito bite. Relapses contribute to increased morbidity, and complicate malaria elimination programs. A single drug effective against hypnozoites, primaquine, is available, but its deployment is curtailed by its haemolytic potential in glucose-6-phosphate dehydrogenase deficient persons. Novel compounds are thus urgently needed to replace primaquine. Discovery of compounds active against hypnozoites is restricted to the in vivo P. cynomolgi-rhesus monkey model. Slow growing hepatic parasites reminiscent of hypnozoites had been noted in cultured P. vivax-infected hepatoma cells, but similar forms are also observed in vitro by other species including P. falciparum that do not produce hypnozoites.Methodology P. falciparum or P. cynomolgi sporozoites were used to infect human or Macaca fascicularis primary hepatocytes, respectively. The susceptibility of the slow and normally growing hepatic forms obtained in vitro to three antimalarial drugs, one active against hepatic forms including hypnozoites and two only against the growing forms, was measured.ResultsThe non-dividing slow growing P. cynomolgi hepatic forms, observed in vitro in primary hepatocytes from the natural host Macaca fascicularis, can be distinguished from similar forms seen in P. falciparum-infected human primary hepatocytes by the differential action of selected anti-malarial drugs. Whereas atovaquone and pyrimethamine are active on all the dividing hepatic forms observed, the P. cynomolgi slow growing forms are highly resistant to treatment by these drugs, but remain susceptible to primaquine.ConclusionResistance of the non-dividing P. cynomolgi forms to atovaquone and pyrimethamine, which do not prevent relapses, strongly suggests that these slow growing forms are hypnozoites. This represents a first step towards the development of a practical medium-throughput in vitro screening assay for novel hypnozoiticidal drugs.

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.

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Plasmodium vivax malaria is characterized by repeated episodes of blood stage infection (relapses) resulting from activation of dormant stages in the liver, so-called hypnozoites. Transition of hypnozoites into developing schizonts has never been observed. A barrier for studying this has been the lack of a system in which to monitor growth of liver stages. Here, exploiting the unique strengths of the simian hypnozoite model P. cynomolgi, we have developed green-fluorescent (GFP) hypnozoites that turn on red-fluorescent (mCherry) upon activation. The transgenic parasites show full liver stage development, including merozoite release and red blood cell infection. We demonstrate that individual hypnozoites actually can activate and resume development after prolonged culture, providing the last missing evidence of the hypnozoite theory of relapse. The few events identified indicate that hypnozoite activation in vitro is infrequent. This system will further our understanding of the mechanisms of hypnozoite activation and may facilitate drug discovery approaches.

Malaria control and elimination are threatened by the emergence and spread of resistance to artemisinin-based combination therapies (ACTs). Experimental evidence suggests that when an artemisinin (ART)-sensitive (K13 wild-type) Plasmodium falciparum strain is exposed to ART derivatives such as dihydroartemisinin (DHA), a small population of the early ring-stage parasites can survive drug treatment by entering cell cycle arrest or dormancy. After drug removal, these parasites can resume growth. Dormancy has been hypothesized to be an adaptive physiological mechanism that has been linked to recrudescence of parasites after monotherapy with ART and, possibly contributes to ART resistance. Here, we evaluate the in vitro drug sensitivity profile of normally-developing P. falciparum ring stages and DHA-pretreated dormant rings (DP-rings) using a panel of antimalarial drugs, including the Plasmodium phosphatidylinositol-4-OH kinase (PI4K)-specific inhibitor KDU691. We report that while KDU691 shows no activity against rings, it is highly inhibitory against DP-rings; a drug effect opposite to that of ART. Moreover, we provide evidence that KDU691 also kills DP-rings of P. falciparum ART-resistant strains expressing mutant K13.

Background The increase in detections of Plasmodium vivax infection in Duffy-negative individuals in Africa has challenged the dogma establishing the unique P. vivax Duffy Binding Protein-Duffy antigen receptor for chemokines (PvDBP-DARC) pathway used by P. vivax merozoites to invade reticulocytes. Information on the impact of Duffy antigen polymorphisms on the epidemiology of P. vivax malaria remains elusive. The objective of this study was to determine the distribution of asexual parasitaemia of P. vivax according to the Duffy antigen polymorphisms in Ethiopia. Methods DNA was extracted from dried blood spots (DBS) collected from prospectively recruited 138 P. vivax-infected patients from health centres. The identification and estimation of P. vivax asexual parasitaemia were performed by microscopic examination and quantitative real-time polymerase chain reaction (PCR). Duffy genotyping was conducted by DNA sequencing in a total of 138 P.vivax infected samples. Results The proportion of Duffy-negatives (FY*BES/FY*BES) in P. vivax infected patients was 2.9% (4/138). Duffy genotype FY*B/FY*BES (48.6%) was the most common, followed by FY*A/FY*BES genotype (25.4%). In one patient, the FY*02 W.01/FY*02 N.01 genotype conferring a weak expression of the Fyb antigen was observed. All P.vivax infected Duffy-negative patients showed low asexual parasitaemia (≤ 110 parasites/µL). The median P. vivax parasitaemia in Duffy-negative patients (53 parasites/µL) was significantly lower than those found in homozygous and heterozygous individuals (P < 0.0001). Conclusion Plasmodium vivax in Duffy-negative patients shows invariably low asexual parasitaemia. This finding suggests that the pathway used by P. vivax to invade Duffy-negative reticulocytes is much less efficient than that used in Duffy-positives. Moreover, the low asexual parasitaemia observed in Duffy-negative individuals could constitute an ‘undetected silent reservoir', thus likely delaying the elimination of vivax malaria in Ethiopia.

Objectives To evaluate Plasmodium malariae susceptibility to current and lead candidate antimalarial drugs. Methods We conducted cross-sectional screening and detection of all Plasmodium species malaria cases, which were nested within a longitudinal prospective study, and an ex vivo assessment of efficacy of a panel of antimalarials against P. malariae and Plasmodium falciparum, both PCR-confirmed mono-infections. Reference compounds tested included chloroquine, lumefantrine, artemether and piperaquine, while candidate antimalarials included the imidazolopiperazine GNF179, a close analogue of KAF156, and the Plasmodium phosphatidylinositol-4-OH kinase (PI4K)-specific inhibitor KDU691. Results We report a high frequency (3%–15%) of P. malariae infections with a significant reduction in ex vivo susceptibility to chloroquine, lumefantrine and artemether, which are the current frontline drugs against P. malariae infections. Unlike these compounds, potent inhibition of P. malariae and P. falciparum was observed with piperaquine exposure. Furthermore, we evaluated advanced lead antimalarial compounds. In this regard, we identified strong inhibition of P. malariae using GNF179, a close analogue of KAF156 imidazolopiperazines, which is a novel class of antimalarial drug currently in clinical Phase IIb testing. Finally, in addition to GNF179, we demonstrated that the Plasmodium PI4K-specific inhibitor KDU691 is highly inhibitory against P. malariae and P. falciparum. Conclusions Our data indicated that chloroquine, lumefantrine and artemether may not be suitable for the treatment of P. malariae infections and the potential of piperaquine, as well as new antimalarials imidazolopiperazines and PI4K-specific inhibitor, for P. malariae cure.