Efficacy of three anti-malarial regimens for uncomplicated Plasmodium falciparum malaria in Cambodia, 2009-2011: a randomized controlled trial and brief review

Abstract

Background: Anti-malarial resistance remains an important public health challenge in Cambodia. The effectiveness of three therapies for uncomplicated falciparum malaria was evaluated in Oddar Meanchey province in Northern Cambodia from 2009 to 2011.

Methods: In this randomized, open-label, parallel group-controlled trial, 211 subjects at least 5 years old with uncomplicated falciparum malaria were treated with 3 days of directly observed therapy: 63 received artesunate-mefloquine (AS/MQ), 77 received dihydroartemisinin-piperaquine (DHA/PPQ), and 71 received atovaquone-proguanil (ATQ/PG). The subjects were followed for 42 days or until recurrent parasitaemia. Genotyping of msp1, msp2, and glurp among individual parasite isolates distinguished recrudescence from reinfection. Pfmdr1 copy number was measured by real-time PCR and half-maximal parasite inhibitory concentrations (IC₅₀) were measured in vitro by 48-h isotopic hypoxanthine incorporation assay.

Results: The per-protocol PCR-adjusted efficacy (95% confidence interval) at 42 days was 80.6% (70.8-90.5%) for AS/MQ, 97.2% (93.3-100%) for DHA/PPQ, and 92.9% (86.1-99.6%) for ATQ/PG. On day 3, 57.9% remained parasitaemic in the AS/MQ and DHA/PPQ arms. At baseline, 46.9% had microscopic Plasmodium falciparum gametocytaemia. Both recurrences in the DHA/PPQ arm lost Pfmdr1 copy number amplification at recrudescence. All four recurrences in the ATQ/PG arm were wild-type for cytochrome bc₁. One subject withdrew from the ATQ/PG arm due to drug allergy.

Conclusions: This study was conducted at the epicentre of substantial multi-drug resistance that emerged soon thereafter. Occurring early in the national transition from AS/MQ to DHA/PPQ, both DHA/PPQ and ATQ/PG had acceptable efficacy against uncomplicated falciparum malaria. However, efficacy of AS/MQ was only 80% with apparent mefloquine resistance based on elevated Pfmdr1 copy number and IC₅₀. By 2009, there was already significant evidence of artemisinin resistance not previously reported at the Northern Cambodia-Thai border. This study suggests the basis for early development of significant DHA/PPQ failures within 3 years of introduction. Artemisinin resistance likely occurred on the Northern border concurrently with that reported along the Western border in Pailin. Trial registration This legacy trial was conducted prior to International Committee of Medical Journal Editors' requirements for preregistration on ClinicalTrials.gov. The full protocol has been provided.

Keywords: Antimalarial resistance; Cambodia; Randomized clinical trial; Therapeutic efficacy; Uncomplicated falciparum malaria.

Fig. 1

Map of Cambodia with Phase 1 and 2 Zones. Site of the 2009 study in relation to previously established malaria containment Zones 1 and 2. Zone 1 was considered the highest risk and had already switched to the use of DHA/PPQ. In addition, there were monitored mass screening and treatment activities using ATQ/PG for PCR-identified subclinical P. falciparum malaria cases

Fig. 2

Consort flow diagram

Fig. 3

Kaplan Meier survival estimates. Kaplan Meier survival analysis was performed for each of the three study groups to compare parasite-free survival between AS/MQ (red), DHA/PPQ (green) and ATQ/PG (blue). Tic marks on each curve indicate a censored subject

Fig. 4

Comparing in vitro parasite drug resistance against common anti-malarials. Samples from 83 subject isolates with > 6400 parasites/µL were run in a classical isotopic P. falciparum in vitro drug resistance assay. In vitro resistance was calculated as the 50% inhibitory concentrations (IC₅₀) based on serial dilutions of artesunate (AS), mefloquine (MQ), dihydroartemisin (DHA), piperaquine (PPQ), chloroquine (CQ) and quinine (QN). Resistance cut-offs established for the assays where known were used (MQ, CQ and QN). Resistance was compared between the three treatment groups (artesunate–mefloquine (A/M) in black circles; dihydroartemisin-piperaquine (DP) in blue squares, and atovaquone–proguanil (AP) in green triangles

Fig. 5

Relationship between mefloquine IC₅₀ and Pfmdr1 status. A Correlation between MQ IC₅₀ and Pfmdr1 copy number. B Mefloquine IC₅₀ was elevated in parasites with ≥ 1.5 copies of Pfmdr1