Marion Barends

Changes in the treatment responses to artesunate-mefloquine on the northwestern border of Thailand during 13 years of continuous deployment

Background Artemisinin combination treatments (ACT) are recommended as first line treatment for falciparum malaria throughout the malaria affected world. We reviewed the efficacy of a 3-day regimen of mefloquine and artesunate regimen (MAS3), over a 13 year period of continuous deployment as first-line treatment in camps for displaced persons and in clinics for migrant population along the Thai-Myanmar border. Methods and Findings 3,264 patients were enrolled in prospective treatment trials between 1995 and 2007 and treated with MAS3. The proportion of patients with parasitaemia persisting on day-2 increased significantly from 4.5% before 2001 to 21.9% since 2002 (p<0.001). Delayed parasite clearance was associated with increased risk of developing gametocytaemia (AOR = 2.29; 95% CI, 2.00–2.69, p = 0.002). Gametocytaemia on admission and carriage also increased over the years (p = 0.001, test for trend, for both). MAS3 efficacy has declined slightly but significantly (Hazards ratio 1.13; 95% CI, 1.07–1.19, p<0.001), although efficacy in 2007 remained well within acceptable limits: 96.5% (95% CI, 91.0–98.7). The in vitro susceptibility of P. falciparum to artesunate increased significantly until 2002, but thereafter declined to levels close to those of 13 years ago (geometric mean in 2007: 4.2 nM/l; 95% CI, 3.2–5.5). The proportion of infections caused by parasites with increased pfmdr1 copy number rose from 30% (12/40) in 1996 to 53% (24/45) in 2006 (p = 0.012, test for trend). Conclusion Artesunate-mefloquine remains a highly efficacious antimalarial treatment in this area despite 13 years of widespread intense deployment, but there is evidence of a modest increase in resistance. Of particular concern is the slowing of parasitological response to artesunate and the associated increase in gametocyte carriage.

Chloroquine Resistant Plasmodium vivax: In Vitro Characterisation and Association with Molecular Polymorphisms

Background Treatment failure of chloroquine for P. vivax infections has reached high levels in the eastern provinces of Indonesia, however, in vitro characterization of chloroquine resistance and its associated molecular profile have yet to be determined. Methods Using a modified schizont maturation assay we investigated the in vitro chloroquine susceptibility profile and molecular polymorphisms of P. vivax isolates collected from Papua, Indonesia, where high levels of clinical chloroquine treatment failure have been reported, and from Thailand, where chloroquine treatment is generally effective. Results The geometric mean chloroquine IC50 for P. vivax isolates from Papua (n = 145) was 312 nM [95%CI: 237–411 nM] compared to 46.8 nM [95%CI: 34.7–63.1 nM] from Thailand (n = 81); p<0.001. Correlating with the known clinical efficacy of the area, a cut off for chloroquine resistance was defined as 220nM, a level exceeded in 13.6% (11/81) of Thai isolates and 65% (94/145) of Papuan isolates; p<0.001. Several sequence polymorphisms in pvcrt-o and pvmdr1, and difference in pvmdr1 copy number were identified. A Y976F mutation in pvmdr1 was present in 96% (123/128) of Papuan isolates and 25% (17/69) of Thai isolates; p<0.001. Overall, the geometric mean chloroquine IC50 in isolates with the Y976F mutation was 283 nM [95%CI: 211–379], compared to 44.5 nM [95%CI: 31.3–63.4] in isolates with the wild type; p< 0.001. Pvmdr1 amplification occurred in 23% (15/66) of Thai isolates compared to none (0/104) of Indonesian isolates (p<0.001), but was not associated with increased chloroquine resistance after controlling for geographical location. Conclusions In vitro susceptibility testing of P. vivax discriminates between populations with differing levels of clinical efficacy of chloroquine. The pvmdr1 polymorphism at Y976F may provide a useful tool to highlight areas of emerging chloroquine resistance, although further studies defining its clinical correlates are needed.

Efficacy and effectiveness of dihydroartemisinin-piperaquine versus artesunate-mefloquine in falciparum malaria: an open-label randomised comparison

BACKGROUND Artemisinin-based combinations are judged the best treatments for multidrug-resistant Plasmodium falciparum malaria. Artesunate-mefloquine is widely recommended in southeast Asia, but its high cost and tolerability profile remain obstacles to widespread deployment. To assess whether dihydroartemisinin-piperaquine is a suitable alternative to artesunate-mefloquine, we compared the safety, tolerability, efficacy, and effectiveness of the two regimens for the treatment of uncomplicated falciparum in western Myanmar (Burma). METHODS We did an open randomised comparison of 3-day regimens of artesunate-mefloquine (12/25 mg/kg) versus dihydroartemisinin-piperaquine (6.3/50 mg/kg) for the treatment of children aged 1 year or older and in adults with uncomplicated falciparum malaria in Rakhine State, western Myanmar. Within each group, patients were randomly assigned supervised or non-supervised treatment. The primary endpoint was the PCR-confirmed parasitological failure rate by day 42. Failure rates at day 42 were estimated by Kaplan-Meier survival analysis. This study is registered as an International Standard Randomised Controlled Trial, number ISRCTN27914471. FINDINGS Of 652 patients enrolled, 327 were assigned dihydroartemisinin-piperaquine (156 supervised and 171 not supervised), and 325 artesunate-mefloquine (162 and 163, respectively). 16 patients were lost to follow-up, and one patient died 22 days after receiving dihydroartemisinin-piperaquine. Recrudescent parasitaemias were confirmed in only two patients; the day 42 failure rate was 0.6% (95% CI 0.2-2.5) for dihydroartemisinin-piperaquine and 0 (0-1.2) for artesunate-mefloquine. Whole-blood piperaquine concentrations at day 7 were similar for patients with observed and non-observed dihydroartemisinin-piperaquine treatment. Gametocytaemia developed more frequently in patients who had received dihydroartemisinin-piperaquine than in those on artesunate-mefloquine: day 7, 18 (10%) of 188 versus five (2%) of 218; relative risk 4.2 (1.6-11.0) p=0.011. INTERPRETATION Dihydroartemisinin-piperaquine is a highly efficacious and inexpensive treatment of multidrug-resistant falciparum malaria and is well tolerated by all age groups. The effectiveness of the unsupervised treatment, as in the usual context of use, equalled its supervised efficacy, indicating good adherence without supervision. Dihydroartemisinin-piperaquine is a good alternative to artesunate-mefloquine.

Adaptive copy number evolution in malaria parasites

Copy number polymorphism (CNP) is ubiquitous in eukaryotic genomes, but the degree to which this reflects the action of positive selection is poorly understood. The first gene in the Plasmodium folate biosynthesis pathway, GTP-cyclohydrolase I (gch1), shows extensive CNP. We provide compelling evidence that gch1 CNP is an adaptive consequence of selection by antifolate drugs, which target enzymes downstream in this pathway. (1) We compared gch1 CNP in parasites from Thailand (strong historical antifolate selection) with those from neighboring Laos (weak antifolate selection). Two percent of chromosomes had amplified copy number in Laos, while 72% carried multiple (2–11) copies in Thailand, and differentiation exceeded that observed at 73 synonymous SNPs. (2) We found five amplicon types containing one to greater than six genes and spanning 1 to >11 kb, consistent with parallel evolution and strong selection for this gene amplification. gch1 was the only gene occurring in all amplicons suggesting that this locus is the target of selection. (3) We observed reduced microsatellite variation and increased linkage disequilibrium (LD) in a 900-kb region flanking gch1 in parasites from Thailand, consistent with rapid recent spread of chromosomes carrying multiple copies of gch1. (4) We found that parasites bearing dhfr-164L, which causes high-level resistance to antifolate drugs, carry significantly (p = 0.00003) higher copy numbers of gch1 than parasites bearing 164I, indicating functional association between genes located on different chromosomes but linked in the same biochemical pathway. These results demonstrate that CNP at gch1 is adaptive and the associations with dhfr-164L strongly suggest a compensatory function. More generally, these data demonstrate how selection affects multiple enzymes in a single biochemical pathway, and suggest that investigation of structural variation may provide a fast-track to locating genes underlying adaptation.

A Randomised Controlled Trial of Artemether-Lumefantrine Versus Artesunate for Uncomplicated Plasmodium falciparum Treatment in Pregnancy

Background To date no comparative trials have been done, to our knowledge, of fixed-dose artemisinin combination therapies (ACTs) for the treatment of Plasmodium falciparum malaria in pregnancy. Evidence on the safety and efficacy of ACTs in pregnancy is needed as these drugs are being used increasingly throughout the malaria-affected world. The objective of this study was to compare the efficacy, tolerability, and safety of artemether-lumefantrine, the most widely used fixed ACT, with 7 d artesunate monotherapy in the second and third trimesters of pregnancy. Methods and Findings An open-label randomised controlled trial comparing directly observed treatment with artemether-lumefantrine 3 d (AL) or artesunate monotherapy 7 d (AS7) was conducted in Karen women in the border area of northwestern Thailand who had uncomplicated P. falciparum malaria in the second and third trimesters of pregnancy. The primary endpoint was efficacy defined as the P. falciparum PCR-adjusted cure rates assessed at delivery or by day 42 if this occurred later than delivery, as estimated by Kaplan-Meier survival analysis. Infants were assessed at birth and followed until 1 y of life. Blood sampling was performed to characterise the pharmacokinetics of lumefantrine in pregnancy. Both regimens were very well tolerated. The cure rates (95% confidence interval) for the intention to treat (ITT) population were: AS7 89.2% (82.3%–96.1%) and AL 82.0% (74.8%–89.3%), p = 0.054 (ITT); and AS7 89.7% (82.6%–96.8%) and AL 81.2% (73.6%–88.8%), p = 0.031 (per-protocol population). One-third of the PCR-confirmed recrudescent cases occurred after 42 d of follow-up. Birth outcomes and infant (up to age 1 y) outcomes did not differ significantly between the two groups. The pharmacokinetic study indicated that low concentrations of artemether and lumefantrine were the main contributors to the poor efficacy of AL. Conclusion The current standard six-dose artemether-lumefantrine regimen was well tolerated and safe in pregnant Karen women with uncomplicated falciparum malaria, but efficacy was inferior to 7 d artesunate monotherapy and was unsatisfactory for general deployment in this geographic area. Reduced efficacy probably results from low drug concentrations in later pregnancy. A longer or more frequent AL dose regimen may be needed to treat pregnant women effectively and should now be evaluated. Parasitological endpoints in clinical trials of any antimalarial drug treatment in pregnancy should be extended to delivery or day 42 if it comes later. Trial Registration: Current Controlled Trials ISRCTN86353884

A Randomized, Controlled Study of a Simple, Once-Daily Regimen of Dihydroartemisinin-Piperaquine for the Treatment of Uncomplicated, Multidrug-Resistant Falciparum Malaria

BACKGROUND Dihydroartemisinin-piperaquine (DP) is a fixed-combination antimalarial drug increasingly deployed in Southeast Asia. The current regimen involves 4 doses given over 3 days. Simplification of the dose regimen should facilitate treatment adherence and thereby increase effectiveness. METHODS In a randomized, controlled, 3-arm trial conducted along the northwestern border of Thailand, the standard 4-dose course of DP (DP4) was compared to an equivalent dose given as a once-daily regimen (DP3) and to the standard treatment of mefloquine-artesunate (MAS3). RESULTS A total of 499 patients were included in the study. Times to fever and parasite clearance were similar in all groups. The PCR genotyping-adjusted cure rates at day 63 after treatment initiation were 95.7% (95% confidence interval [95% CI], 92.2%-98.9%) for MAS3, 100% for DP4, and 99.4% (95% CI, 98.1%-100%) for DP3. The DP4 and DP3 cure rates were significantly higher than that for MAS3 (P=.008 and P=.03, respectively). All regimens were well tolerated. There were 3 deaths (1 in the MAS3 group and 2 in the DP3 group), all of which were considered to be unrelated to treatment. Rates of other adverse events were comparable between the groups, except for diarrhea, which was more common in the DP4 group (P=.05 vs. the MAS3 group). CONCLUSIONS A once-daily, 3-dose regimen of DP is a highly efficacious treatment for multidrug-resistant falciparum malaria. This simple, safe, and relatively inexpensive fixed combination could become the treatment of choice for falciparum malaria.

Amplification of pvmdr1 associated with multidrug-resistant Plasmodium vivax.

BACKGROUND Multidrug-resistant strains of Plasmodium vivax are emerging in Southeast Asia. METHODS In vitro drug susceptibility and pvmdr1 genotype were determined in P. vivax field isolates from Indonesia and Thailand. RESULTS Increased pvmdr1 copy number was present in 21% of isolates from Thailand (15/71) and none from Indonesia (0/114; P < .001). Compared with Indonesian isolates, the median IC(50) of Thai isolates was lower for chloroquine (36 vs. 114 nmol/L; P < .001) but higher for amodiaquine (34 vs. 13.7 nmol/L; P = .032), artesunate (8.33 vs. 1.58 nmol/L; P < .001), and mefloquine (111 vs. 9.87 nmol/L; P < .001). In 11 cryopreserved Thai isolates, those with increased pvmdr1 copy number had a higher IC(50) for mefloquine (78.6 vs. 38 nmol/L for single-copy isolates; P = .006). Compared with isolates with the wild-type allele, the Y976F mutation of pvmdr1 was associated with reduced susceptibility to chloroquine (154 nmol/L [range, 4.6-3505] vs. 34 nmol/L [range, 6.7-149]; P < .001) but greater susceptibility to artesunate (1.8 vs. 9.5 nmol/L; P = .009) and mefloquine (14 vs. 121 nmol/L; P < .001). CONCLUSIONS Amplification of pvmdr1 and single-nucleotide polymorphisms are correlated with susceptibility of P. vivax to multiple antimalarial drugs. Chloroquine and mefloquine appear to exert competitive evolutionary pressure on pvmdr1, similar to that observed with pfmdr1 in Plasmodium falciparum.

A Randomized Comparison of Artesunate-Atovaquone-Proguanil versus Quinine in Treatment for Uncomplicated Falciparum Malaria during Pregnancy

BACKGROUND There is no safe, practical, and effective treatment for pregnant women infected with multidrug-resistant Plasmodium falciparum. METHODS We recruited pregnant Karen women in the second or third trimesters of pregnancy who had uncomplicated falciparum malaria for a randomized, open-label trial with a restricted sequential trial design of 7 days of supervised quinine (SQ7) versus 3 days of artesunate-atovaquone-proguanil (AAP). RESULTS Eight-one pregnant women entered the study between December 2001 and July 2003; 42 were treated with SQ7 and 39 were treated with AAP. Fever, parasite clearance, and duration of anemia were significantly better with AAP; the treatment failure rate was 7 times lower (5% [2/39] vs. 37% [15/41]; relative risk, 7.1 [95% confidence interval, 1.7-29.2]; P = .001). There were no significant differences in birth weight, duration of gestation, or congenital abnormality rates in newborns or in growth and developmental parameters of infants monitored for 1 year. CONCLUSION AAP is a well-tolerated, effective, practical, but expensive treatment for multidrug-resistant falciparum malaria during the second or third trimesters of pregnancy. Despite the small number of subjects, our results add to the growing body of evidence that AAP is safe for the mother and the fetus.

Randomized Comparison of Chloroquine plus Sulfadoxine-Pyrimethamine versus Artesunate plus Mefloquine versus Artemether-Lumefantrine in the Treatment of Uncomplicated Falciparum Malaria in the Lao People's Democratic Republic

BACKGROUND Recent clinical trials in the Lao Peoples Democratic Republic have demonstrated that chloroquine and sulfadoxine-pyrimethamine, which are national malaria treatment policy, are no longer effective in the treatment of uncomplicated Plasmodium falciparum malaria. METHODS A randomized comparison of 3 oral antimalarial combinations--chloroquine plus sulfadoxine-pyrimethamine versus artesunate plus mefloquine versus artemether-lumefantrine--with 42-day follow-up period, was conducted among 330 patients with acute uncomplicated falciparum malaria in southern Laos. RESULTS The 42-day cure rates, as determined by intention-to-treat analysis and adjusted for reinfection, were 100%, 97%, and 93% for the groups receiving artesunate plus mefloquine, artemether-lumefantrine, and chloroquine plus sulfadoxine-pyrimethamine, respectively. Of 8 patients receiving chloroquine plus sulfadoxine-pyrimethamine who experienced treatment failure, 6 had early treatment failure. The mean parasite clearance time was significantly longer in patients treated with chloroquine plus sulfadoxine-pyrimethamine (2.9 days; 95% confidence interval [CI], 2.8-3.0 days) than in those treated with artesunate plus mefloquine (2.07 days; 95% CI, 2.0-2.1 days; P<.001) and artemether-lumefantrine (2.08 days; 95% CI, 2.0-2.1 days; P<.001). Cure rates with artemether-lumefantrine were high despite low mean daily dietary fat intake (13.8 g; 95% CI, 12.5-15.1 g) and day 7 plasma lumefantrine concentrations (0.47 mu g/mL; 95% CI, 0.38-0.56 mu g/mL). CONCLUSION Oral artesunate plus mefloquine and artemether-lumefantrine are highly effective for the treatment of uncomplicated falciparum malaria in Laos.

An open, randomized comparison of artesunate plus mefloquine vs. dihydroartemisinin–piperaquine for the treatment of uncomplicated Plasmodium falciparum malaria in the Lao People's Democratic Republic (Laos)

Objective  To determine the efficacy and safety of oral dihydroartemisinin–piperaquine (DP, ArtekinTM) in the treatment of uncomplicated Plasmodium falciparum malaria in southern Laos.