Clinical and In Vitro Resistance of Plasmodium falciparum to Artesunate-Amodiaquine in Cambodia

Abstract

Background: Artesunate-amodiaquine is a potential therapy for uncomplicated malaria in Cambodia.

Methods: Between September 2016 and January 2017, artesunate-amodiaquine efficacy and safety were evaluated in a prospective, open-label, single-arm observational study at health centers in Mondulkiri, Pursat, and Siem Reap Provinces, Cambodia. Adults and children with microscopically confirmed Plasmodium falciparum malaria received oral artesunate-amodiaquine once daily for 3 days plus single-dose primaquine, with follow-up on days 7, 14, 21, and 28. The primary outcome was day-28 polymerase chain reaction (PCR)-adjusted adequate clinical and parasitological response (ACPR). An amodiaquine parasite survival assay (AQSA) was developed and applied to whole genome sequencing results to evaluate potential amodiaquine resistance molecular markers.

Results: In 63 patients, day-28 PCR-adjusted ACPR was 81.0% (95% confidence interval [CI], 68.9-88.7). Day 3 parasite positivity rate was 44.4% (28/63; 95% CI, 31.9-57.5). All 63 isolates had the K13(C580Y) marker for artemisinin resistance; 79.4% (50/63) had Pfpm2 amplification. The AQSA resistance phenotype (≥45% parasite survival) was expressed in 36.5% (23/63) of isolates and was significantly associated with treatment failure (P = .0020). Pfmdr1 mutant haplotypes were N86/184F/D1246, and Pfcrt was CVIET or CVIDT at positions 72-76. Additional Pfcrt mutations were not associated with amodiaquine resistance, but the G353V mutant allele was associated with ACPR compared to Pfmdr1 haplotypes harboring F1068L or S784L/R945P mutations (P = .030 and P = .0004, respectively).

Conclusions: For uncomplicated falciparum malaria in Cambodia, artesunate-amodiaquine had inadequate efficacy owing to amodiaquine-resistant P. falciparum. Amodiaquine resistance was not associated with previously identified molecular markers.

Keywords: Plasmodium falciparum; Cambodia; artemisinin; artesunate-amodiaquine; drug resistance.

Figure 1.

Kaplan-Meier estimates of ACPR with artesunate-amodiaquine for uncomplicated malaria. There were no reinfections on PCR-adjustment and no patients were censored. Abbreviations: ACPR, adequate clinical and parasitological response; CI, confidence interval; PCR, polymerase chain reaction.

Figure 2.

Relationship between mono-desethyl-amodiaquine IC₅₀ determined in the [³H]-hypoxanthine uptake inhibition assay and clinical outcome at day 28 following treatment with artesunate-amodiaquine for 63 P. falciparum clinical isolates. Open circles represent P. falciparum isolates and black horizontal bars and Ι bars indicate the median and interquartile range, statistical comparison used the Mann-Whitney test. Abbreviations: ACPR, adequate clinical and parasitological response; IC_50, half-maximal inhibitory concentration.

Figure 3.

Parasite survival in the amodiaquine survival assay (AQSA). A, Correlation between AQSA parasite survival and IC₅₀ determined in the [³H]-hypoxanthine uptake inhibition assay. B, Relationship between AQSA parasite survival and clinical outcome at day 28 following treatment with artesunate-amodiaquine. Open circles represent P. falciparum isolates and black horizontal bars and Ι bars indicate the median and interquartile range, statistical comparison used the Mann-Whitney U test. C, Kaplan-Meier estimates of ACPR for parasites with the AQSA resistance phenotype (≥45% survival) versus those with the susceptible phenotype (<45% survival), statistical comparison used the log-rank test (Mantel-Cox). Abbreviations: ACPR, adequate clinical and parasitological response; IC_50, half-maximal inhibitory concentration.

Figure 4.

Effect of P. falciparum Pfcrt–Pfmdr1 haplotype on resistance phenotype. A, Parasite survival in the amodiaquine survival assay (AQSA) for Cambodian clinical isolates (N = 92), the horizontal gray dotted line indicates the AQSA resistance phenotype (≥45% parasite survival). Symbols represent P. falciparum isolates, and black horizontal bars and Ι bars indicate the median and interquartile range; statistical comparison used the Kruskal-Wallis test. B, Kaplan-Meier plots for day-28 ACPR in 60 patients with P. falciparum malaria treated with artesunate-amodiaquine for amodiaquine-resistant and -susceptible Pfcrt–Pfmdr1 haplotypes. Abbreviation: ACPR, adequate clinical and parasitological response.