Therapeutic and Transmission-Blocking  Efficacy of Dihydroartemisinin/Piperaquine and Chloroquine against Plasmodium vivax Malaria, Cambodia

We assessed the efficacy of standard 3-day courses of chloroquine and dihydroartemisinin/piperaquine against Plasmodium vivax malaria. Compared with chloroquine, dihydroartemisinin/piperaquine was faster in clearing asexual P. vivax parasites and blocking human-to-mosquito transmission. This drug combination was also more effective in preventing potential recurrences for >2 months.


The Study
We conducted an open-label, randomized, control trial in June-December 2014 in Rattanakiri Province, Cambodia. Febrile patients or patients with a history of fever in the previous 48 h who sought treatment in health facilities and had positive results by rapid diagnostic test (CareStart Malaria HRP2/pLDH Pf/PAN Combo; Access Bio, Inc., Somerset, NJ, USA) for non-P. falciparum malaria were offered participation in the study. Pregnant or lactating women and patients with signs of severe malaria, other known illnesses, or inability to provide informed consent were excluded. Patients with P. vivax monoinfection confirmed by PCR were eligible for the study (5).
At enrollment, after we obtained written informed consent, patients were randomized to receive supervised standard 3-day courses of DHA/PPQ (Duo-Cotecxin; Zhejiang Holley Nanhu Pharmaceutical Co., Ltd., Jiaxing, China) or chloroquine (Nivaquine; Sanofi-Aventis, Paris, France). For each participant, medical histories were obtained and clinical and biological examinations performed. We followed up with patients according to an extended World Health Organization protocol on days 1, 2, 3, 5, and 7 and then weekly until day 63. At each visit, we performed clinical examinations and obtained an axillary temperature and a capillary blood sample.
Malaria parasites were detected by microscopy (Giemsastained blood films) and PCR as described (5). Chloroquine resistance was ruled out for patients if no parasites were detected by microscopy on day 28 or, in case of recurrence, if the chloroquine blood concentration on the day of recurrence did not exceed >100 ng/mL (6,7). We measured chloroquine blood concentrations by using liquid chromatography-tandem mass spectrometry for 50-μL samples of whole blood.
During January-March 2016, we conducted an additional study at the same site to evaluate the infectivity of P. vivax from blood of symptomatic patients to Anopheles dirus mosquito vectors; we tested pretreatment and posttreatment blood samples by using membrane feeding assays without serum replacement (8). Any febrile patients seeking antimalarial treatment with similar inclusion/exclusion criteria described previously were enrolled in this study.
After we obtained written informed consent, we fed batches of 50 An. dirus mosquitoes with blood collected from these patients on 3 occasions: 1) before the first dose of DHA/PPQ or chloroquine, 2) on the same day at 9:00 pm (i.e., 2-11 h after treatment), and 3) at 24 h posttreatment for patients treated with chloroquine. We performed statistical analyses by using GraphPad Prism 5 (GraphPad, San Diego,
For the drug comparison study, we enrolled 50 patients (25 in each study arm); in each arm, 5 patients were lost to follow-up during days 2-35. A total of 40 patients (20 in each  (10). ¶Mosquitoes were 6-8 days old and were allowed to feed for 20 min. Feeding was conducted at the same place immediately after obtaining blood from patients. Dissections were performed 6 days after the blood meal. Midguts were dissected in 1% mercurochrome stain, and the presence and number of oocysts were determined by microscopy (×20 magnification). #Patients were defined as being infectious when >1 mosquito became infected with oocysts. study arm) were followed up until day 63. Baseline patient characteristics were similar for both patient groups (Table  1). We did not observe any adverse events or early clinical failures. The proportion of patients still parasitemic on days 1 and 2 (detected by microscopy) was lower for the DHA/ PPQ-treated group than for the chloroquine-treated group ( Table 2). Medians of the parasite reduction ratio recorded on days 1 and 2 were higher for the DHA/PPQ-treated patient group (Table 2). All patients, regardless of their treatment, were microscopically parasite free at day 3. Within 2 months of follow up, there were fewer patients with a recurrence (detected by PCR) in the DHA/PPQtreated group than in the chloroquine-treated group (odds ratio 0.17, 95% CI 0.05-0.66; p<0.05, by log-rank test, p<0.01 by Kaplan-Meier survival analysis) (Table 2; Figure  1). Median time to recurrence after treatments was also delayed in patients given DHA/PPQ (56 days) compared with those patients given chloroquine (49 days) ( Table  2). No recurrence occurred before day 28 in either study arm, which is suggestive of relapse or reinfection, rather than recrudescence of drug-resistant parasites (6,7). In the chloroquine-treated group, 12/20 patients with recurrence had a chloroquine blood concentration <100 ng/mL on the day of recurrence (chloroquine + desethyl chloroquine: median 55.6 ng/mL, interquartile range 40.0-61.7 ng/mL); these results excluded likely chloroquine resistance (6,7).
For the mosquito-to-human transmission study, we enrolled 19 patients (9 given chloroquine and 10 given DHA/PPQ). Baseline patient characteristics were similar in both patient groups, except for day 0 parasitemia and gametocytemia, which were higher for the DHA/PPQtreated group ( Table 1). The proportion of infectious blood from P. vivax-infected patients and the median proportion of infected mosquitoes fed on blood collected before the  Each dot represents the parasite transmissibility reduction ratio (i.e., 100 -[proportion of infected mosquitoes fed with blood samples collected at 9:00 pm after the first dose of treatment × 100/ proportion of infected mosquitoes fed with blood samples collected at patient enrollment before the first dose of treatment]). Only data of infectious patients at enrollment are shown (17/19 patients; Table  1). For the chloroquine-treated patient group, a second mosquito blood feeding was performed 24 h after the first dose. DHA/PPQ, dihydroartemisinin/piperaquine.

Plasmodium vivax Malaria, Cambodia
first dose of DHA/PPQ or chloroquine were similar for both patient groups (Table 1). Despite an initial higher day 0 gametocytemia for the DHA/PPQ-treated group, the proportions of infectious P. vivax blood collected at 9:00 pm after the first dose of DHA/PPQ or chloroquine and the median proportion of infected mosquitoes were lower for the DHA/PPQ-treated group than for the chloroquinetreated group (Table 2). Overall, DHA/PPQ acted faster than chloroquine in decreasing over time the proportion of infectious patients (generalized linear mixed model time for drug interaction, χ 1 2 = 113.1, p <0.0001) (Figure 2). For the group given chloroquine, 2 (22%) of 9 blood samples were still infectious 24 hours after the first dose ( Table 2).

Conclusions
We confirm that DHA/PPQ acts faster (<48 h) than chloroquine (≈72 h) in eliminating sexual and asexual P. vivax parasites and that DHA/PPQ provides an excellent postexposure prophylaxis against potential recurrences for >2 months (11). This benefit relies on the combination of artemisinin derivatives (DHA), which are fast-acting drugs capable of eliminating any P. vivax blood stages, and a long-lasting partner drug (PPQ), which has a long terminal elimination half-life and is highly effective in preventing P. vivax recurrence for up to 56 days. Although the number of patients enrolled was small, we demonstrated that DHA/PPQ also acts faster (<5 h) than chloroquine in killing P. vivax sexual stages and thus prevents the risk for transmission of parasites to the mosquito vector the night after uptake of the first dose. This rapid clearance of gametocytes is a major benefit of DHA/PPQ in comparison with chloroquine, given that P. vivax gametocytes appear early in the course of disease and must be eliminated as soon as possible to limit risk of transmission (12,13).
In summary, our findings support the recommendation of DHA/PPQ as first-line treatment for P. falciparum and P. vivax uncomplicated malaria in regions to which these species are co-endemic. These findings apply to areas in which chloroquine is still effective and no P. falciparum resistance to PPQ has been observed.