Reduced artemisinin susceptibility of Plasmodium falciparum ring stages in western Cambodia

Abstract

The declining efficacy of artemisinin derivatives against Plasmodium falciparum in western Cambodia is a major concern. The knowledge gap in the understanding of the mechanisms involved hampers designing monitoring tools. Here, we culture-adapted 20 isolates from Pailin and Ratanakiri (areas of artemisinin resistance and susceptibility in western and eastern Cambodia, respectively) and studied their in vitro response to dihydroartemisinin. No significant difference between the two sets of isolates was observed in the classical isotopic test. However, a 6-h pulse exposure to 700 nM dihydroartemisinin (ring-stage survival assay -RSA]) revealed a clear-cut geographic dichotomy. The survival rate of exposed ring-stage parasites (ring stages) was 17-fold higher in isolates from Pailin (median, 13.5%) than in those from Ratanakiri (median, 0.8%), while exposed mature stages were equally and highly susceptible (0.6% and 0.7%, respectively). Ring stages survived drug exposure by cell cycle arrest and resumed growth upon drug withdrawal. The reduced susceptibility to artemisinin in Pailin appears to be associated with an altered in vitro phenotype of ring stages from Pailin in the RSA.

Fig 1

Schematic representation of the various in vitro phenotypes investigated with synchronous cultures. RSA, ring-stage survival assay; MSA, mature-stage survival assay; SIA, standard in vitro drug susceptibility assay; RA, recovery assay: RGAA, ring-stage growth arrest assay.

Fig 2

In vitro susceptibility to eight antimalarial drugs (expressed as IC₅₀) of 20 adapted parasites collected in Pailin (n = 10) and Ratanakiri (n = 10) in 2010 to 2011 determined using the standard growth inhibition assay (SIA). PL, Pailin; RTK, Ratanakiri. Median IC₅₀s: for artesunate (AS), 0.61 nM for PL and 0.38 nM for RTK (P = 0.26); for dihydroartemisinin (DHA), 0.47 nM for PL and 0.32 nM for RTK (P = 0.07); for atovaquone (ATV), 6.9 nM for PL and 11.2 nM for RTK (P = 0.02); for doxycycline (DOX), 5.7 μM for PL for PL and 4.8 μM for RTK (P = 0.22); for mefloquine (MQ), 30.4 nM for PL and 31.6 nM for RTK (P = 0.60); for chloroquine (CQ), 170 nM for PL and 69 nM for RTK (P = 0.01); for piperaquine (PIP), 37.6 nM for PL and 33.2 nM for RTK (P = 0.10); and for quinine (QN), 198 nM for PL and 160 nM for RTK (P = 0.25). The quality control data determined using 3D7 were as follows (median IC₅₀ ± SD): 0.7 nM ± 0.3 nM (n = 21) for AS, 0.5 nM ± 0.2 nM (n = 21) for DHA, 19 nM ± 9 nM (n = 22) for ATV, 5.21 μM ± 1.8 nM (n = 21) for DOX, 24 nM ± 9 nM (n = 21) for MQ, 16 nM ± 4 nM (n = 22) for CQ, 36 nM ± 5 nM (n = 22) for PIP, and 74 nM ± 32 nM (N=22) for ON.

Fig 3

Results of the RSA and MSA expressed as the percentage of viable parasites following a 6-h exposure to 700 nM DHA of ring- and mature-stage parasites from Pailin and Ratanakiri and reference clones (W2, 3D7, HB3, G15, and 7G8).

Fig 4

Results of the recovery assay (RA) following a 6-h exposure to 700 nM DHA of ring-stage parasites in separate and mixed cultures. (A) Recovery of the initial parasitemia for PL4971 from Pailin (red), RT4974 from Ratanakiri (green), and a mixed culture with an initial 1:1 ratio (orange). Recovery of the initial parasitemia from D2 to D6 of separate cultures (for PL4971, 5%, 30%, 63%, 133%, and 132.5%; for RT4974, 0.45%, 0.3%, 1.4%, 1.9%, and 5.5%) and mixed cultures (2.35%, 14.5%, 26%, 60.4%, and 72.5%) that were perfectly matched. (B) Relative expression of msp-1 alleles (Ct_Mad20 for PL4971 and Ct_K1 for RT4974) in exposed cultures (red) and nonexposed cultures (blue).

Fig 5

Recrudescence following a 24-h exposure to 700 nM DHA of ring stages with or without sorbitol treatment after drug exposure. (A) Parasitemia of control cultures (no drug exposure) of PL4971 (red) and RT4974 (blue). Cultures received a sorbitol treatment at H24 (S+) (dotted line) or were mock treated (S−) (solid line). Sorbitol treatment induced, as predicted, massive lysis of infected red cells and a dramatic drop of parasite counts (at D6, PL4971 = 14.3% in S− versus 0.2% in S+ and RT4974 = 5.6% in S− versus 0.3% in S+). (B) Recovery of the initial parasitemia of DHA-exposed PL4971 (red), PL5145 (green), PL4992 (purple), RT3592 (brown), RT4974 (blue), and RT5150 (black). Cultures received a sorbitol treatment immediately after a 24-h exposure to 700 nM DHA (S+) (dotted line) or were mock treated (S−) (solid line). In DHA-treated cultures, sorbitol treatment had no impact on the growth curve of cultures (at D10, PL4971 = 77.7% in S− versus 70.6% in S+, PL5145 = 47.8% in S− versus 49.5% in S+, PL4992 = 14.5% in S− versus 11.5% in S+, RT3592= 10.6% in S− versus 11.6% in S+, RT4974 = 3.5% in S− versus 3.9% in S+, and RT5150 = 2.5% in S− versus 2.3% in S+).

Fig 6

Morphology of PL5145 parasites from H0 to H72 in nonexposed cultures and exposed cultures (24-h exposure to 700 nM DHA). The figure shows Giemsa-stained smears (100× magnification).