Altered drug susceptibility during host adaptation of a <i>Plasmodium falciparum</i> strain in a non-human primate model

Infections with Plasmodium falciparum, the most pathogenic of the Plasmodium species affecting man, have been reduced in part due to artemisinin-based combination therapies. However, artemisinin resistant parasites have recently emerged in South-East Asia. Novel intervention strategies are therefore urgently needed to maintain the current momentum for control and elimination of this disease. In the present study we characterize the phenotypic and genetic properties of the multi drug resistant (MDR) P. falciparum Thai C2A parasite strain in the non-human Aotus primate model, and across multiple passages. Aotus infections with C2A failed to clear upon oral artesunate and mefloquine treatment alone or in combination, and ex vivo drug assays demonstrated reduction in drug susceptibility profiles in later Aotus passages. Further analysis revealed mutations in the pfcrt and pfdhfr loci and increased parasite multiplication rate (PMR) across passages, despite elevated pfmdr1 copy number. Altogether our experiments suggest alterations in parasite population structure and increased fitness during Aotus adaptation. We also present data of early treatment failures with an oral artemisinin combination therapy in a pre-artemisinin resistant P. falciparum Thai isolate in this animal model

Altered drug susceptibility during host adaptation of a Plasmodium falciparum strain in a non-human primate model

Infections with Plasmodium falciparum, the most pathogenic of the Plasmodium species affecting man, have been reduced in part due to artemisinin-based combination therapies. However, artemisinin resistant parasites have recently emerged in South-East Asia. Novel intervention strategies are therefore urgently needed to maintain the current momentum for control and elimination of this disease. In the present study we characterize the phenotypic and genetic properties of the multi drug resistant (MDR) P. falciparum Thai C2A parasite strain in the non-human Aotus primate model, and across multiple passages. Aotus infections with C2A failed to clear upon oral artesunate and mefloquine treatment alone or in combination, and ex vivo drug assays demonstrated reduction in drug susceptibility profiles in later Aotus passages. Further analysis revealed mutations in the pfcrt and pfdhfr loci and increased parasite multiplication rate (PMR) across passages, despite elevated pfmdr1 copy number. Altogether our experiments suggest alterations in parasite population structure and increased fitness during Aotus adaptation. We also present data of early treatment failures with an oral artemisinin combination therapy in a pre-artemisinin resistant P. falciparum Thai isolate in this animal model

Insights into an Optimization of <i>Plasmodium vivax</i> Sal-1 <i>In Vitro</i> Culture: The <i>Aotus</i> Primate Model

<div><p>Malaria is one of the most significant tropical diseases, and of the <i>Plasmodium</i> species that cause human malaria, <i>P</i>. <i>vivax</i> is the most geographically widespread. However, <i>P</i>. <i>vivax</i> remains a relatively neglected human parasite since research is typically limited to laboratories with direct access to parasite isolates from endemic field settings or from non-human primate models. This restricted research capacity is in large part due to the lack of a continuous <i>P</i>. <i>vivax in vitro</i> culture system, which has hampered the ability for experimental research needed to gain biological knowledge and develop new therapies. Consequently, efforts to establish a long-term <i>P</i>. <i>vivax</i> culture system are confounded by our poor knowledge of the preferred host cell and essential nutrients needed for <i>in vitro</i> propagation. Reliance on very heterogeneous <i>P</i>. <i>vivax</i> field isolates makes it difficult to benchmark parasite characteristics and further complicates development of a robust and reliable culture method. In an effort to eliminate parasite variability as a complication, we used a well-defined <i>Aotus</i>-adapted <i>P</i>. <i>vivax</i> Sal-1 strain to empirically evaluate different short-term <i>in vitro</i> culture conditions and compare them with previous reported attempts at <i>P</i>. <i>vivax in vitro</i> culture Most importantly, we suggest that reticulocyte enrichment methods affect invasion efficiency and we identify stabilized forms of nutrients that appear beneficial for parasite growth, indicating that <i>P</i>. <i>vivax</i> may be extremely sensitive to waste products. Leuko-depletion methods did not significantly affect parasite development. Formatting changes such as shaking and static cultures did not seem to have a major impact while; in contrast, the starting haematocrit affected both parasite invasion and growth. These results support the continued use of <i>Aotus</i>-adapted Sal-1 for development of <i>P</i>. <i>vivax</i> laboratory methods; however, further experiments are needed to optimize culture conditions to support long-term parasite development.</p></div

Shaking and static format support cultures similarly.

<p>(<b>A</b>) Glass T flask used for shaking conditions to replicate the conditions used in [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004870#pntd.0004870.ref014" target="_blank">14</a>]. (<b>B</b>) Parasitemias of two biological replicates (MN23026b and MN23009b) grown in shaking or static conditions at equal haematocrits. (<b>C</b>) Conversion rates between rings to trophozoites or trophozoites to schizonts in the two biological replicates (<b>D</b>) Parasitized erythrocyte multiplication rate (PEMR) of the two biological replicates in either shaking or static conditions using reticulocytes from hemochromatosis patients enriched by density. Error bars represent the standard error. Results are not statically significant.</p

Reticulocyte enrichment method can affect invasion of <i>P</i>. <i>vivax</i> Sal-1.

<p>(<b>A</b>) Table showing methods used to enrich reticulocytes from peripheral blood derived either from hemochromatosis patients and Buffy Coat blood packs. The enrichment was performed according to either published studies or manufacturer’s instructions detailed in the Materials and Methods. The resulting total reticulocyte percentage was determined by staining with NMB. (<b>B</b>) The final fold change in parasitemia following invasion can be normalized to compare different invasion assays using the parasitized erythrocyte multiplication rate (PEMR) by dividing the final number of rings by the input number of schizonts [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004870#pntd.0004870.ref018" target="_blank">18</a>]. (<b>C</b>) PEMR demonstrates that the hemochromatosis reticulocytes enriched by density using the [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004870#pntd.0004870.ref014" target="_blank">14</a>] method resulted in the best invasion in two independent biological replicates. This invasion was better than the invasion in the same blood enriched by Percoll and significantly better than the reticulocytes from Buffy Coat enriched by density or CD71-microbeads. Parasites were from <i>Aotus</i> MN21014 and MN28016. Statistical significance was determined using Dunnett’s multiple comparison test. P value <0.05 (<b>D</b>) Final ring parasitemias of the invasion assays in B. Error bars represent the standard error.</p

Starting haematocrit (hct) can influence <i>in vitro</i> outcome of <i>P</i>.<i>vivax</i> Sal-1.

<p>Cultures from two independent biological replicates, MN23009a and MN23009b, were initiated at different hcts. Reticulocytes enriched by density from patients with hemochromatosis were used. Cultures were initiated with the initial draw at 6% or 12% hct (iRBC, infected RBC) or were initiated with the 6% initial draw (iRBC) and hct was doubled to 12% hct with uninfected enriched reticulocytes (density hemochromatosis) (12% iRBC + RBC) to account for any differences due to the overall total parasite biomass. (<b>A</b>). Parasitemia over time from the two biological replicates. Survival was affected by the conversion differences in B as the two cultures were initiated at majority rings (MN23009a) or mixed rings and trophozoites (MN23009b) (<b>B</b>) Conversion rate differences in the two biological replicates in A between rings to trophozoites and trophozoites to schizonts. (<b>C</b>) Parasitized erythrocyte multiplication rate was only possible to measure in one biological replicate, MN23009a. Error bars represent the standard error. To set up a culture at 12% hct, we first set up the culture at 6% hct and doubled the hct with uninfected fresh, hemochromatosis human blood (12% iRBC + RBC). Results were not statically significant.</p

Media supplementation can influence the health and growth of the parasite.

<p>(<b>A</b>) Parasitemia graph showing the effect of GlutaMAX on parasitemia at different time points using three independent biologicals <i>P</i>. <i>vivax</i> Sal-1 parasites from MN23062a and b and MN23009a. Reticulocytes from hemochromatosis patients enriched by density were used for these experiments. (<b>B</b>) Histogram comparing parasite stages at different time points from the parasites in A: (<b>C</b>) Conversion percentages of rings to trophozoites and trophozoites to schizonts in media supplemented with or without GlutaMAX. Data from the three independent biological replicates in A. Error bars represent the standard error. While the parasitemia differences are not statically significant, we did observed a longer persistence of parasites in the cultures supplemented with GlutaMAX and we did observed reinvasion only in the GlutaMAX culture.</p

Experimental design using <i>Aotus lemurinus lemurinus</i> monkeys.

<p>Diagram showing the inoculations of the <i>Aotus</i> monkeys with <i>P</i>. <i>vivax</i> Sal-1. The percentages shown are for staging of the parasites at bleed (R, rings; T, trophozoites; S, schizonts; G, gametocytes). Table shows the experiments that each bleed was used for.</p