Volume 23, Number 4—April 2017
Treatment Failure of Dihydroartemisinin/Piperaquine for Plasmodium falciparum Malaria, Vietnam
The high failure rate of artemisinin-based combination therapy in the treatment of uncomplicated Plasmodium falciparum malaria has been a growing concern in the Greater Mekong Subregion of Southeast Asia. High numbers of treatment failures were reported for dihydroartemisinin/piperaquine in Cambodia (1), but efficacy of this drug has remained high in Vietnam since its introduction in 2003. We investigated dihydroartemisinin/piperaquine efficacy in the treatment of uncomplicated P. falciparum malaria in Binh Phuoc Province, Vietnam, during August–December 2015. We looked for molecular markers of drug resistance and determined piperaquine blood levels in treated patients to assess if piperaquine resistance was present in Vietnam. This study was approved by the ethics boards of the Ministry of Health in Vietnam and the Western Pacific Regional Office of the World Health Organization.
The National Institute of Malariology, Parasitology, and Entomology conducted this study as part of routine surveillance on drug efficacy following the 2009 World Health Organization protocol (2). After obtaining written consent, patients (age of inclusion, 2–60 years) were enrolled and given dihydroartemisinin/piperaquine (Pharbaco, Hanoi, Vietnam) at a target dosage of 2.4 mg/kg for dihydroartemisinin and 18 mg/kg for piperaquine once a day for 3 days. Patients with treatment failures were subsequently given quinine hydrochlorate (30 mg/kg/d) and doxycycline (3 mg/kg/d) for 7 days. Primary endpoint was adequate clinical and parasitologic response (ACPR) on day 42; PCR genotyping, comparing day 0 and day of failure samples, was used to distinguish recrudescence from reinfection with another strain (2). Dried blood spots were collected on day 0 and analyzed for mutations in the K13 propeller domain (3), Pfmdr1 copy number (4), and Pfplasmepsin2 (PfPM2) copy number (5), which are markers associated with artemisinin, mefloquine, and piperaquine resistance, respectively. By using a previously established relationship between capillary whole blood and venous plasma, piperaquine plasma concentrations were calculated from blood spots collected day 7 (6). Sequencing was done by the Institut Pasteur in Cambodia, and the piperaquine blood levels were assessed by the Mahidol Oxford Tropical Medicine Research Unit in Thailand.
Forty-six patients with uncomplicated P. falciparum malaria were enrolled; 44 were followed until day 42, and 2 were lost to follow-up after day 14. Mean age of enrolled patients was 26.9 (range 14–53) years, and 93% (43/46) were male. Geometric mean parasitemia on day 0 was 17,759 (range 1,514–97,454)/μL.
On day 3, half (23/46) of patients were parasitemic. On day 42, a total of 65% (26/40, 95% CI 48.3%–79.4%) had an ACPR, and 35% (14/40) had recrudescence; 4 were withdrawn because they became reinfected.
Artemisinin resistance is defined as delayed parasite clearance and is associated with mutations in the K13 propeller domain, the most prevalent being the C580Y mutation in the eastern Greater Mekong Subregion (7). K13 analysis of 42 samples (4 were excluded because of uninterpretable results) from our study showed that 90.5% (38/42) were C580Y and 9.5% (4/42) were wild-type. This C580Y prevalence is higher than that reported in a previous study done in Binh Phuoc in 2014, in which 34.5% of samples had the C580Y mutation (B.Q. Phuc, unpub. data).
Analysis of PfPM2 showed that 25/46 (54.3%) samples had multiple copies of the gene. Of the 42 samples with known K13 types, 22 (52.4%) had both C580Y and PfPM2 amplifications. The remaining 3 had unknown K13 types. All 46 samples had a single copy of Pfmdr1, indicating that all parasites were sensitive to mefloquine (4).
The average day 7 piperaquine plasma concentration (n = 42) was 35.7 (range 11.1–71.0) ng/mL. In 57.1% (24/42) of patients, this concentration was at or above the cutoff value (30 ng/mL) associated with adequate piperaquine exposure (1). For patients with ACPRs, the average concentration was 36.9 (range 17.2–71.0) ng/mL, and 57.7% (15/26) were adequately exposed. For patients that had recrudescence, the average concentration was 39.5 (range 12.4–65.7) ng/mL, and 72.7% (8/11) were adequately exposed.
Of the 14 patients who experienced recrudescence, 10 had parasites with the C580Y mutation and PfPM2 amplifications, 3 had parasites with the C580Y mutation only, and 1 had parasites with an unknown K13 type and PfPM2 amplifications. K13 mutations (found during routine surveillance conducted over the last 5 years in Vietnam) alone did not lead to dihydroartemisinin/piperaquine failures. The association between the presence of molecular markers and recrudescence is confounded by various factors, including parasite load, immunity, and drug levels. Of the 3 patients who had recrudescence and were infected with P. falciparum without PfPM2 amplifications, 2 had inadequate piperaquine levels. Of the 11 patients who had recrudescence and an infection with P. falciparum with PfPM2 amplifications, 7 had adequate piperaquine levels. Low piperaquine blood levels, irrespective of the presence of PfPM2 amplifications, might play a role in some treatment failures. Treatment failures in cases with PfPM2 amplification–positive parasites and adequate piperaquine exposure support the presence of piperaquine resistance in Vietnam.
Our results show that 1 K13 mutation has become dominant and that piperaquine resistance is present in Vietnam. A change in the malaria treatment policy to treat with artesunate/mefloquine in Binh Phuoc Province is underway.
Dr. Phuc is an associate professor and the Chief of the Department of Clinical Research, National Institute of Malariology, Parasitology, and Entomology, Hanoi, Vietnam. His primary research interests are clinical trials and drug efficacy in malaria.
The authors would like to express their appreciation to all of the study patients.
This study was supported by the Bill and Melinda Gates Foundation and the US Agency for International Development via the World Health Organization.
The authors declare that there is no conflict of interest regarding the publication of this paper. D.B., C.R., G.G., and P.R. are staff members of the World Health Organization. D.B., C.R., G.G., and P.R. are solely responsible for views expressed in this publication, and they do not necessarily represent decisions, policies, or views of the World Health Organization.
- Leang R, Taylor WR, Bouth DM, Song L, Tarning J, Char MC, et al. Evidence of Plasmodium falciparum malaria multidrug resistance to artemisinin and piperaquine in western Cambodia: dihydroartemisinin-piperaquine open-label multicenter clinical assessment. Antimicrob Agents Chemother. 2015;59:4719–26.
- World Health Organization. Methods for surveillance of antimalarial drug efficacy. Geneva: The World Health Organization; 2009.
- Ariey F, Witkowski B, Amaratunga C, Beghain J, Langlois A-C, Khim N, et al. A molecular marker of artemisinin-resistant Plasmodium falciparum malaria. Nature. 2014;505:50–5.
- Sidhu AB, Uhlemann AC, Valderramos SG, Valderramos JC, Krishna S, Fidock DA. Decreasing pfmdr1 copy number in plasmodium falciparum malaria heightens susceptibility to mefloquine, lumefantrine, halofantrine, quinine, and artemisinin. J Infect Dis. 2006;194:528–35.
- Witkowski B, Duru V, Khim N, Ross LS, Saintpierre B, Beghain J, et al. A surrogate marker of piperaquine-resistant Plasmodium falciparum malaria: a phenotype-genotype association study. Lancet Infect Dis. 2016;S1473-3099(16)30415-7.
- Ashley EA, Stepniewska K, Lindegardh N, Annerberg A, Tarning J, McGready R, et al. Comparison of plasma, venous and capillary blood levels of piperaquine in patients with uncomplicated falciparum malaria. Eur J Clin Pharmacol. 2010;66:705–12.
- Ménard D, Khim N, Beghain J, Adegnika AA, Shafiul-Alam M, Amodu O, et al.; KARMA Consortium. A worldwide map of Plasmodium falciparum K13-propeller polymorphisms. N Engl J Med. 2016;374:2453–64.