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

Abstract

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.

Figure 1. Antimalarial drug responses of Plasmodium falciparum C2A infected Aotus monkeys.

(A) Parasitemia response plots of Aotus monkeys infected with the P. falciparum C2A clone passage X. Animals were inoculated with 5 × 10⁶ infected red blood cells from a donor monkey (MN26006, passage IX) and treated when parasitemia reached >10⁵ parasites × μL of blood. Panel (A) shows group 1 animals treated with MQ at 40 mg/Kg orally once, group 2 animals treated with AS at 33 mg/Kg orally × three days and group 3 animals treated with MQ and AS at 33 mg/Kg × three days and MQ at 40 mg/Kg orally once. Panel (B) shows a spleen intact control treated with AS at 33 mg/Kg × three days +MQ at 40 mg/Kg orally once on day 16 of infection. Panel (C) shows parasitemia responses of MN24058 and MN26008 inoculated with PfC2A passages III and IV respectively and treated with AS at 33 mg/Kg × three days i.v. +MQ at 40 mg/Kg orally once.

Figure 2. Ex vivo/in vitro antimalarial drug susceptibility and Pfmdr1 copy number variation of PfC2A across Aotus passages.

(A,B) IC₅₀ antimalarial drug nM concentration bar plots of reference strains D6 (white bar), in vitro culture adapted TM90C2A (blue bar) and pfC2A Aotus adapted passage levels II (black bar) and X (red bar). Drug IC50 nM resistance threshold (black line). Mean ± sample SEM (see also Table S1). (C) DHA drug susceptibility based on RSA. Survival percentage = DHA treated parasites/DMSO treated parasites × 100. Red bar represents survival of artemisinin resistant positive control strain KH001-029, D6 is included as negative reference strain. Significant survival threshold (black line) is set at 1% as in Witkowski et al.. (D) Passage level pfmdr1 copy number fold change of a Plasmodium falciparum C2A clone during adaption to Aotus monkeys. P. falciparum strains with two pfmdr1 gene copies are considered resistant to MQ. Mean ± sample SEM. Dashed line indicates threshold for pfmdr1 CN level indicative of MQ resistance. Dotted line indicates threshold for pfmdr1 CN level indicative of MQ sensitivity. MQ = Mefloquine; CQ = Chloroquine; ATV = Atovaquone; AS = Artesunate; QHS = Artemisinin; DHA = Dihydroartemisinin. p = Mann-Whitney U significance t test unpaired samples. ***p < 0.005; **p < 0.05.

Figure 3. Molecular barcode and antimalarial drug resistance loci across C2A passages.

(A) Plasmodium falciparum barcode. (B) Drug resistance genotyping profiles. nd = not determined; λ = spleen intact.

Figure 4. Plasmodium falciparum C2A growth across Aotus passages.

(A) Parasitemia plots from individual monkeys (colored lines) with time of treatment (black dotted lines) across each passage. Parasitemia was followed for up to 100 days post inoculation and frequently fell below detectability (disappearance of colored lines). (B) Day of peak parasitemia trend. The day of peak parasitemia is the time since inoculation when peak parasitemia was achieved, averaged across monkeys for each passage. (C) Peak parasitemia trend. The value of peak parasitemia is the maximum parasitemia reached over the course of infection, averaged across monkeys for each passage. (D) Parasite multiplication rate (PMR). The PMR is the median of parasitemia increases across 48-hr periods over the course of an infection, averaged across monkeys for each passage. The data is modeled with linear regression.

Figure 5. Genealogy of Plasmodium. falciparum C2A adaptation to Aotus lemurinus lemurinus monkeys.

Blue highlighted squares indicate samples from which gDNA was extracted for genetic studies. Aotus monkey numbers in bold mark those that have been used in this study.