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

Comparison of PvLAP5 and Pvs25 qRT-PCR assays for the detection of Plasmodium vivax gametocytes in field samples preserved at ambient temperature from remote malaria endemic regions of Panama

Background: As the elimination of malaria in Mesoamerica progresses, detection of Plasmodium vivax using light microscopy (LM) becomes more difficult. Highly sensitive molecular tools have been developed to help determine the hidden reservoir of malaria transmission in low transmission settings. In this study we compare the performance of PvLAP5 and Pvs25 qRT-PCR assays to LM for the detection of Plasmodium vivax gametocytes in field samples preserved at ambient temperature from malaria endemic regions of Panama. Methods: For this purpose, we collected a total of 83 malaria field samples during 2017-2020 preserved in RNAprotect (RNAp) of which 63 (76%) were confirmed P. vivax by LM and selected for further analysis. Additionally, 16 blood samples from local healthy malaria smear negative volunteers, as well as, from 15 malaria naïve lab-bred Aotus monkeys were used as controls. To optimize the assays, we first determined the minimum blood volume sufficient for detection of PvLAP5 and Pv18SrRNA using P. vivax infected Aotus blood that was preserved in RNAp and kept either at ambient temperature for up to 8 days before freezing or was snap-frozen at -80° Celsius at the time of bleeding. We then compared the mean differences in gametocyte detection rates of both qRT-PCR assays to LM and performed a multivariate correlation analysis of study variables. Finally, we determined the sensitivity (Se) and specificity (Sp) of the assays at detecting gametocytes compared to LM. Results: Blood volume optimization indicated that a blood volume of at least 60 μL was sufficient for detection of PvLAP5 and Pv18SrRNA and no significant differences were found between RNA storage conditions. Both PvLAP5 and Pvs25 qRT-PCR assays showed a 37-39% increase in gametocyte detection rate compared to LM respectively. Strong positive correlations were found between gametocytemia and parasitemia and both PvLAP5 and Pvs25 gametocyte markers. However, no significant differences were detected in the Se and Sp of the Pvs25 and PvLAP5 qRT-PCR assays, even though data from control samples suggested Pvs25 to be more abundant than PvLAP5. Conclusions: This study shows that the PvLAP5 qRT-PCR assay is as Se and Sp as the gold standard Pvs25 assay and is at least 37% more sensitive than LM at detecting P. vivax gametocytes in field samples preserved in RNAp at ambient temperature from malaria endemic regions of Panama. Author summary: Plasmodium vivax is one of the five species of malaria (P. falciparum, P. malariae, P. ovale and P. knowlesi) that are transmitted to man by the bite of female anopheles mosquitoes. It causes ~14.3 million cases mainly in Southeast Asia, India, the Western Pacific and the Americas annually. In the Americas, malaria remains a major problem in underdeveloped areas and indigenous communities in the Amazon region and eastern Panama, where it is endemic and difficult to eliminate. As malaria elimination progresses, detection of P. vivax by light microscopy (LM) becomes more difficult. Therefore, highly sensitive molecular tools have been developed that use genetic markers for the parasite to help determine the hidden reservoir of malaria transmission. This study compares the performance of two molecular assays based on the genetic markers of mature gametocytes PvLAP5 and Pvs25 with LM. The study shows that the PvLAP5 qRT-PCR assay is as sensitive and specific as the gold standard Pvs25 assay and is at least 37% more sensitive than LM at detecting P. vivax gametocytes. These data suggest that the PvLAP5 qRT-PCR assay can be a useful tool to help determine the hidden reservoir of transmission in endemic foci approaching elimination

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

Malaria is one of the most significant tropical diseases, and of the Plasmodium species that cause human malaria, P. vivax is the most geographically widespread. However, P. vivax 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 P. vivax in vitro 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 P. vivax culture system are confounded by our poor knowledge of the preferred host cell and essential nutrients needed for in vitro propagation. Reliance on very heterogeneous P. vivax 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 Aotus-adapted P. vivax Sal-1 strain to empirically evaluate different short-term in vitro culture conditions and compare them with previous reported attempts at P. vivax in vitro 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 P. vivax 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 Aotus-adapted Sal-1 for development of P. vivax laboratory methods; however, further experiments are needed to optimize culture conditions to support long-term parasite development

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