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Concerns about malaria parasite resistance to treatment with artemisinin drugs (ARTs) have grown with findings of prolonged parasite clearance t 1/2 s (>5 h) and their association with mutations in Plasmodium falciparum Kelch-propeller protein K13. Here, we describe a P. falciparum laboratory cross of K13 C580Y mutant with C580 wild-type parasites to investigate ART response phenotypes in vitro and in vivo. After genotyping >400 isolated progeny, we evaluated 20 recombinants in vitro: IC 50 measurements of dihydroartemisinin were at similar low nanomolar levels for C580Y- and C580-type progeny (mean ratio, 1.00; 95% CI, 0.62-1.61), whereas, in a ring-stage survival assay, the C580Y-type progeny had 19.6-fold (95% CI, 9.76-39.2) higher average counts. In splenectomized Aotus monkeys treated with three daily doses of i.v. artesunate, t 1/2 calculations by three different methods yielded mean differences of 0.01 h (95% CI, -3.66 to 3.67), 0.80 h (95% CI, -0.92 to 2.53), and 2.07 h (95% CI, 0.77-3.36) between C580Y and C580 infections. Incidences of recrudescence were 57% in C580Y (4 of 7) versus 70% in C580 (7 of 10) infections (-13% difference; 95% CI, -58% to 35%). Allelic substitution of C580 in a C580Y-containing progeny clone (76H10) yielded a transformant (76H10 C580Rev ) that, in an infected monkey, recrudesced regularly 13 times over 500 d. Frequent recrudescences of ART-treated P. falciparum infections occur with or without K13 mutations and emphasize the need for improved partner drugs to effectively eliminate the parasites that persist through the ART component of combination therapy., Competing Interests: Conflict of interest statement: R.M.F. and D.E.G. are coauthors of a paper published in 2017. N.F.G., D.A.F., and D.E.G. are coauthors of papers published in 2016 and 2018. K.M., C.A.L., and D.E.G. are coauthors of a paper published in 2014. R.S.R. and S.R.M. are coauthors of papers published in 2015 and 2017.

Plasmodium vivax causes heavy burdens of disease across malarious regions worldwide. Mature P. vivax asexual and transmissive gametocyte stages occur in the blood circulation, and it is often assumed that accumulation/sequestration in tissues is not an important phase in their development. Here, we present a systematic study of P. vivax stage distributions in infected tissues of nonhuman primate (NHP) malaria models as well as in blood from human infections. In a comparative analysis of the transcriptomes of P. vivax and Plasmodium falciparum blood-stage parasites, we found a conserved cascade of stage-specific gene expression despite the greatly different gametocyte maturity times of these two species. Using this knowledge, we validated a set of conserved asexual- and gametocyte-stage markers both by quantitative real-time PCR and by antibody assays of peripheral blood samples from infected patients and NHP ( Aotus sp.). Histological analyses of P. vivax parasites in organs of 13 infected NHP ( Aotus and Saimiri species) demonstrated a major fraction of immature gametocytes in the parenchyma of the bone marrow, while asexual schizont forms were enriched to a somewhat lesser extent in this region of the bone marrow as well as in sinusoids of the liver. These findings suggest that the bone marrow is an important reservoir for gametocyte development and proliferation of malaria parasites. IMPORTANCE Plasmodium vivax malaria continues to cause major public health burdens worldwide. Yet, significant knowledge gaps in the basic biology and epidemiology of P. vivax malaria remain, largely due to limited available tools for research and diagnostics. Here, we present a systematic examination of tissue sequestration during P. vivax infection. Studies of nonhuman primates and malaria patients revealed enrichment of developing sexual stages (gametocytes) and mature replicative stages (schizonts) in the bone marrow and liver, relative to those present in peripheral blood. Identification of the bone marrow as a major P. vivax tissue reservoir has important implications for parasite diagnosis and treatment., (Copyright © 2018 Obaldia et al.)

Background: Artemisinin-based combination therapy is recommended to treat Plasmodium falciparum worldwide, but observations of longer artemisinin (ART) parasite clearance times (PCTs) in Southeast Asia are widely interpreted as a sign of potential ART resistance. In search of an in vitro correlate of in vivo PCT after ART treatment, a ring-stage survival assay (RSA) of 0-3 h parasites was developed and linked to polymorphisms in the Kelch propeller protein (K13). However, RSA remains a laborious process, involving heparin, Percoll gradient, and sorbitol treatments to obtain rings in the 0-3 h window. Here two alternative RSA protocols are presented and compared to the standard Percoll-based method, one highly stage-specific and one streamlined for laboratory application., Methods: For all protocols, P. falciparum cultures were synchronized with 5 % sorbitol treatment twice over two intra-erythrocytic cycles. For a filtration-based RSA, late-stage schizonts were passed through a 1.2 μm filter to isolate merozoites, which were incubated with uninfected erythrocytes for 45 min. The erythrocytes were then washed to remove lysis products and further incubated until 3 h post-filtration. Parasites were pulsed with either 0.1 % dimethyl sulfoxide (DMSO) or 700 nM dihydroartemisinin in 0.1 % DMSO for 6 h, washed twice in drug-free media, and incubated for 66-90 h, when survival was assessed by microscopy. For a sorbitol-only RSA, synchronized young (0-3 h) rings were treated with 5 % sorbitol once more prior to the assay and adjusted to 1 % parasitaemia. The drug pulse, incubation, and survival assessment were as described above., Results: Ring-stage survival of P. falciparum parasites containing either the K13 C580 or C580Y polymorphism (associated with low and high RSA survival, respectively) were assessed by the described filtration and sorbitol-only methods and produced comparable results to the reported Percoll gradient RSA. Advantages of both new methods include: fewer reagents, decreased time investment, and fewer procedural steps, with enhanced stage-specificity conferred by the filtration method., Conclusions: Assessing P. falciparum ART sensitivity in vitro via RSA can be streamlined and accurately evaluated in the laboratory by filtration or sorbitol synchronization methods, thus increasing the accessibility of the assay to research groups.