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Artemisinin-resistant Plasmodium falciparum in Pursat province, western Cambodia: a parasite clearance rate study

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Summary

Background

Artemisinin-resistant Plasmodium falciparum has been reported in Pailin, western Cambodia, detected as a slow parasite clearance rate in vivo. Emergence of this phenotype in western Thailand and possibly elsewhere threatens to compromise the effectiveness of all artemisinin-based combination therapies. Parasite genetics is associated with parasite clearance rate but does not account for all variation. We investigated contributions of both parasite genetics and host factors to the artemisinin-resistance phenotype in Pursat, western Cambodia.

Methods

Between June 19 and Nov 28, 2009, and June 26 and Dec 6, 2010, we enrolled patients aged 10 years or older with uncomplicated falciparum malaria, a density of asexual parasites of at least 10 000 per μL of whole blood, no symptoms or signs of severe malaria, no other cause of febrile illness, and no chronic illness. We gave participants 4 mg/kg artesunate at 0, 24, and 48 h, 15 mg/kg mefloquine at 72 h, and 10 mg/kg mefloquine at 96 h. We assessed parasite density on thick blood films every 6 h until undetectable. The parasite clearance half-life was calculated from the parasite clearance curve. We genotyped parasites with 18 microsatellite markers and patients for haemoglobin E, α-thalassaemia, and a mutation of G6PD, which encodes glucose-6-phosphate dehydrogenase. To account for the possible effects of acquired immunity on half-life, we used three surrogates for increased likelihood of exposure to P falciparum: age, sex, and place of residence. This study is registered with ClinicalTrials.gov, number NCT00341003.

Findings

We assessed 3504 individuals from all six districts of Pursat province seeking treatment for malaria symptoms. We enrolled 168 patients with falciparum malaria who met inclusion criteria. The geometric mean half-life was 5·85 h (95% CI 5·54–6·18) in Pursat, similar to that reported in Pailin (p=0·109). We identified two genetically different parasite clone groups: parasite group 1 (PG1) and parasite group 2 (PG2). Non-significant increases in parasite clearance half-life were seen in patients with haemoglobin E (0·55 h; p=0·078), those of male sex (0·96 h; p=0·064), and in 2010 (0·68 h; p=0·068); PG1 was associated with a significant increase (0·79 h; p=0·033). The mean parasite heritability of half-life was 0·40 (SD 0·17).

Interpretation

Heritable artemisinin resistance is established in a second Cambodian province. To accurately identify parasites that are intrinsically susceptible or resistant to artemisinins, future studies should explore the effect of erythrocyte polymorphisms and specific immune responses on half-life variation.

Funding

Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health.

Introduction

Artemisinin resistance in Plasmodium falciparum has not yet been fully defined. In Pailin, western Cambodia, it has been described as a slower parasite clearance rate after artesunate treatment than in Wang Pha, western Thailand.1 A 2012 report2 showed that artemisinin resistance is emerging in western Thailand, and predicts that in 2–6 years it will reach levels reported in Pailin. In Cambodia, the provinces of Pailin, Battambang, Pursat, and Kampot constitute zone 1—a region where the global plan for artemisinin resistance containment focuses its parasite elimination efforts because persistent parasitaemia has been reported more than 72 h after artemisinin-based combination therapy.3 However, parasite clearance rates in zone 1 provinces other than Pailin (population 70 482 in 2008;4 area 803 km2) have not been reported.

In Pailin and western Thailand, the finding that genetically related P falciparum clones are cleared at similar rates in different patients suggests that parasite genetics has a role.2, 5 However, much variation in parasite clearance rates remains unexplained, implying that host factors affect this phenotype. Erythrocyte polymorphisms are common in western Cambodia, where greater than 40% of individuals carry at least the haemoglobin E (HbE) polymorphism.6, 7

Erythrocyte polymorphisms and acquired immunity could account for some variation in parasite clearance rates, thus affecting association analyses between these rates and parasite genotypes. One possibility is that oxidative stress exerted by HbE,8 α-thalassaemia,9 and erythrocytes deficient in glucose-6-phosphate dehydrogenase (G6PD)10 stimulates ring-stage parasites to upregulate their antioxidant defences, thereby antagonising the pro-oxidant activity of artemisinins11 and delaying parasite clearance by the spleen.12, 13, 14 Another possibility is that effectors of acquired immunity (eg, immune IgG) remove parasites from the circulation,15 thereby accelerating parasite clearance. To assess the effects of parasite genetics and host factors on the artemisinin-resistance phenotype in western Cambodia, we studied parasite clearance rates in response to artesunate in Pursat province (population 397 107 in 2008;4 area 12 692 km2; 146 km south of Pailin).

Section snippets

Study design and participants

During the 2009 and 2010 malaria seasons (June 19 to Nov 28, 2009; June 26 to Dec 6, 2010) we did a study at the Sampov Meas Referral Hospital in Pursat, Cambodia. We enrolled patients from all six districts of Pursat province. Patients were referred to our study clinic from Sampov Meas, Krakor, Bakan, and Kandieng districts. Our study team recruited patients from Kravanh and Veal Veng districts by travelling daily through these westernmost regions to assess patients presenting at the side of

Results

We assessed 3504 individuals from all six districts of Pursat province seeking treatment for malaria symptoms. 712 people (20·3%) were diagnosed with falciparum malaria, but 544 (76·4%) of these patients were not enrolled because of low parasite densities or other exclusion criteria (appendix). The remaining 168 patients were predominantly men with a median age of 24 years (table), which is consistent with our previous findings (unpublished). 103 patients (61·3%) had at least one erythrocyte

Discussion

The time taken to clear P falciparum parasites in patients from Pursat is lengthy, with half-life as long as 11·28 h, suggesting that the artemisinin-resistance phenotype is well established outside Pailin (panel; appendix). We have identified a genetically defined cluster of parasites (PG1) that is significantly associated with increased half-life; a heritability analysis suggested that roughly 40% of half-life variation can be attributed to parasite genetics. Additionally, some host factors

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