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1.  Multiple populations of artemisinin-resistant Plasmodium falciparum in Cambodia 
Nature genetics  2013;45(6):10.1038/ng.2624.
We describe an analysis of genome variation in 825 Plasmodium falciparum samples from Asia and Africa that reveals an unusual pattern of parasite population structure at the epicentre of artemisinin resistance in western Cambodia. Within this relatively small geographical area we have discovered several distinct but apparently sympatric parasite subpopulations with extremely high levels of genetic differentiation. Of particular interest are three subpopulations, all associated with clinical resistance to artemisinin, which have skewed allele frequency spectra and remarkably high levels of haplotype homozygosity, indicative of founder effects and recent population expansion. We provide a catalogue of SNPs that show high levels of differentiation in the artemisinin-resistant subpopulations, including codon variants in various transporter proteins and DNA mismatch repair proteins. These data provide a population genetic framework for investigating the biological origins of artemisinin resistance and for defining molecular markers to assist its elimination.
doi:10.1038/ng.2624
PMCID: PMC3807790  PMID: 23624527
3.  Reduced Artemisinin Susceptibility of Plasmodium falciparum Ring Stages in Western Cambodia 
The declining efficacy of artemisinin derivatives against Plasmodium falciparum in western Cambodia is a major concern. The knowledge gap in the understanding of the mechanisms involved hampers designing monitoring tools. Here, we culture-adapted 20 isolates from Pailin and Ratanakiri (areas of artemisinin resistance and susceptibility in western and eastern Cambodia, respectively) and studied their in vitro response to dihydroartemisinin. No significant difference between the two sets of isolates was observed in the classical isotopic test. However, a 6-h pulse exposure to 700 nM dihydroartemisinin (ring-stage survival assay -RSA]) revealed a clear-cut geographic dichotomy. The survival rate of exposed ring-stage parasites (ring stages) was 17-fold higher in isolates from Pailin (median, 13.5%) than in those from Ratanakiri (median, 0.8%), while exposed mature stages were equally and highly susceptible (0.6% and 0.7%, respectively). Ring stages survived drug exposure by cell cycle arrest and resumed growth upon drug withdrawal. The reduced susceptibility to artemisinin in Pailin appears to be associated with an altered in vitro phenotype of ring stages from Pailin in the RSA.
doi:10.1128/AAC.01868-12
PMCID: PMC3553720  PMID: 23208708
4.  Efficacy of Dihydroartemisinin-Piperaquine for Treatment of Uncomplicated Plasmodium falciparum and Plasmodium vivax in Cambodia, 2008 to 2010 
We describe here the results of antimalarial therapeutic efficacy studies conducted in Cambodia from 2008 to 2010. A total of 15 studies in four sentinel sites were conducted using dihydroartemisinin-piperaquine (DP) for the treatment of Plasmodium falciparum infection and chloroquine (CQ) and DP for the treatment of P. vivax infection. All studies were performed according to the standard World Health Organization protocol for the assessment of antimalarial treatment efficacy. Among the studies of DP for the treatment of P. falciparum, an increase in treatment failure was observed in the western provinces. In 2010, the PCR-corrected treatment failure rates for DP on day 42 were 25% (95% confidence interval [CI] = 10 to 51%) in Pailin and 10.7% (95% CI = 4 to 23%) in Pursat, while the therapeutic efficacy of DP remained high (100%) in Ratanakiri and Preah Vihear provinces, located in northern and eastern Cambodia. For the studies of P. vivax, the day 28 uncorrected treatment failure rate among patients treated with CQ ranged from 4.4 to 17.4%; DP remained 100% effective in all sites. Further study is required to investigate suspected P. falciparum resistance to piperaquine in western Cambodia; the results of in vitro and molecular studies were not found to support the therapeutic efficacy findings. The emergence of artemisinin resistance in this region has likely put additional pressure on piperaquine. Although DP appears to be an appropriate new first-line treatment for P. vivax in Cambodia, alternative treatments are urgently needed for P. falciparum-infected patients in western Cambodia.
doi:10.1128/AAC.00686-12
PMCID: PMC3553743  PMID: 23208711
5.  Pharmacokinetics and Pharmacodynamics of Oral Artesunate Monotherapy in Patients with Uncomplicated Plasmodium falciparum Malaria in Western Cambodia 
Antimicrobial Agents and Chemotherapy  2012;56(11):5484-5493.
Artemisinin-resistant malaria along the Thailand-Cambodian border is an important public health concern, yet mechanisms of drug action and their contributions to the development of resistance are poorly understood. The pharmacokinetics and pharmacodynamics of oral artesunate monotherapy were explored in a dose-ranging trial in an area of emerging artesunate resistance in western Cambodia. We enrolled 143 evaluable subjects with uncomplicated Plasmodium falciparum malaria in an open label study of directly observed artesunate monotherapy at 3 dose levels (2, 4, and 6 mg/kg of body weight/day) for 7 days at Tasanh Health Center, Tasanh, Cambodia. Clinical outcomes were similar among the 3 groups. Wide variability in artesunate and dihydroartemisinin concentrations in plasma was observed. No significant dose-effect or concentration-effect relationships between pharmacokinetic (PK) and parasite clearance parameters were observed, though baseline parasitemia was modestly correlated with increased parasite clearance times. The overall parasite clearance times were prolonged compared with the clearance times in a previous study at this site in 2006 to 2007, but this did not persist when the evaluation was limited to subjects with a comparable artesunate dose (4 mg/kg/day) and baseline parasitemia from the two studies. Reduced plasma drug levels with higher presentation parasitemias, previously hypothesized to result from partitioning into infected red blood cells, was not observed in this population with uncomplicated malaria. Neither in vitro parasite susceptibility nor plasma drug concentrations appeared to have a direct relationship with the pharmacodynamic (PD) effects of oral artesunate on malaria parasites. While direct concentration-effect relationships were not found, it remains possible that a population PK modeling approach that allows modeling of greater dose separation might discern more-subtle relationships.
doi:10.1128/AAC.00044-12
PMCID: PMC3486599  PMID: 22869581
6.  Reduced Susceptibility of Plasmodium falciparum to Artesunate in Southern Myanmar 
PLoS ONE  2013;8(3):e57689.
Background
Plasmodium falciparum resistance to artemisinins, the first line treatment for malaria worldwide, has been reported in western Cambodia. Resistance is characterized by significantly delayed clearance of parasites following artemisinin treatment. Artemisinin resistance has not previously been reported in Myanmar, which has the highest falciparum malaria burden among Southeast Asian countries.
Methods
A non-randomized, single-arm, open-label clinical trial of artesunate monotherapy (4 mg/kg daily for seven days) was conducted in adults with acute blood-smear positive P. falciparum malaria in Kawthaung, southern Myanmar. Parasite density was measured every 12 hours until two consecutive negative smears were obtained. Participants were followed weekly at the study clinic for three additional weeks. Co-primary endpoints included parasite clearance time (the time required for complete clearance of initial parasitemia), parasite clearance half-life (the time required for parasitemia to decrease by 50% based on the linear portion of the parasite clearance slope), and detectable parasitemia 72 hours after commencement of artesunate treatment. Drug pharmacokinetics were measured to rule out delayed clearance due to suboptimal drug levels.
Results
The median (range) parasite clearance half-life and time were 4.8 (2.1–9.7) and 60 (24–96) hours, respectively. The frequency distributions of parasite clearance half-life and time were bimodal, with very slow parasite clearance characteristic of the slowest-clearing Cambodian parasites (half-life longer than 6.2 hours) in approximately 1/3 of infections. Fourteen of 52 participants (26.9%) had a measurable parasitemia 72 hours after initiating artesunate treatment. Parasite clearance was not associated with drug pharmacokinetics.
Conclusions
A subset of P. falciparum infections in southern Myanmar displayed markedly delayed clearance following artemisinin treatment, suggesting either emergence of artemisinin resistance in southern Myanmar or spread to this location from its site of origin in western Cambodia. Resistance containment efforts are underway in Myanmar.
Trial Registration
Australian New Zealand Clinical Trials Registry ACTRN12610000896077
doi:10.1371/journal.pone.0057689
PMCID: PMC3592920  PMID: 23520478
7.  Efficacy of Artemether-Lumefantrine in Area of High Malaria Endemicity in India and Its Correlation with Blood Concentration of Lumefantrine 
This study was conducted to correlate blood concentrations of lumefantrine with treatment outcome for patients with Plasmodium falciparum malaria when the drug was given without specific instructions for administration with food. Patients with P. falciparum malaria in the highly endemic state of Orissa, India, were enrolled during 2008 and followed-up for 28 days after admistration of artemether-lumefantrine for three days according to a World Health Organization protocol. Drug concentration in whole blood was determined by using blood spots placed on filter paper on day 7. The technology is suitable for field studies. One hundred percent of the patients had an adequate clinical and parasitological response. These results confirm the efficacy of artemether-lumefantrine in persons from poor tribal communities when given without specific instructions regarding co-administration with food, despite high inter-individual variability in blood concentrations of lumefantrine.
doi:10.4269/ajtmh.2012.11-0182
PMCID: PMC3284351  PMID: 22403306
8.  Current Status of Artemisinin-Resistant falciparum Malaria in South Asia: A Randomized Controlled Artesunate Monotherapy Trial in Bangladesh 
PLoS ONE  2012;7(12):e52236.
Objective
Recent reports indicate that first cases of genuine artemisinin resistance have already emerged along the Thai-Cambodian border. The main objective of this trial was to track the potential emergence of artemisinin resistance in Bangladesh, which in terms of drug resistance forms a gateway to the Indian subcontinent.
Methods
We conducted an open-label, randomized, controlled 42-day clinical trial in Southeastern Bangladesh to investigate the potential spread of clinical artemisinin resistance from Southeast Asia. A total of 126 uncomplicated falciparum malaria patients were randomized to one of 3 treatment arms (artesunate monotherapy with 2 or 4 mg/kg/day once daily or quinine plus doxycycline TID for 7 days). Only cases fulfilling a stringent set of criteria were considered as being artemisinin-resistant.
Findings
The 28-day and 42-day cure rates in the artesunate monotherapy (2 and 4 mg/kg) and quinine/doxycyline arms were 97.8% (95% confidence interval, CI: 87.8–99.8%), 100% (95% CI: 91.1–100%), and 100% (95% CI: 83.4–100%), respectively. One case of re-infection was seen in the artesunate high dose arm, and a single case of recrudescence was observed in the low dose group on day 26. No differences in median parasite and fever clearance times were found between the 2 artesunate arms (29.8 h and 17.9 h vs. 29.5 h and 19.1 h). Not a single case fulfilled our criteria of artemisinin resistance. Parasite clearance times were considerably shorter and ex vivo results indicate significantly higher susceptibility (50% inhibitory concentration for dihydroartemisinin was 1.10 nM; 95% CI: 0.95–1.28 nM) to artemisinins as compared to SE-Asia.
Conclusion
There is currently no indication that artemisinin resistance has reached Bangladesh. However, the fact that resistance has recently been reported from nearby Myanmar indicates an urgent need for close monitoring of artemisinin resistance in the region.
Trial Registration
ClinicalTrials.gov NCT00639873.
doi:10.1371/journal.pone.0052236
PMCID: PMC3525560  PMID: 23272227
9.  Effect of High-Dose or Split-Dose Artesunate on Parasite Clearance in Artemisinin-Resistant Falciparum Malaria 
New treatment strategies are needed for artemisinin-resistant falciparum malaria. This randomized trial shows that neither increasing nor splitting the standard once-daily artesunate dose reverses the markedly reduced parasite clearance rate in patients with artemisinin-resistant falciparum malaria.
Background. The emergence of Plasmodium falciparum resistance to artemisinins on the Cambodian and Myanmar-Thai borders poses severe threats to malaria control. We investigated whether increasing or splitting the dose of the short-half-life drug artesunate improves parasite clearance in falciparum malaria in the 2 regions.
Methods. In Pailin, western Cambodia (from 2008 to 2010), and Wang Pha, northwestern Thailand (2009–2010), patients with uncomplicated falciparum malaria were randomized to oral artesunate 6 mg/kg/d as a once-daily or twice-daily dose for 7 days, or artesunate 8 mg/kg/d as a once-daily or twice-daily dose for 3 days, followed by mefloquine. Parasite clearance and recrudescence for up to 63 days of follow-up were assessed.
Results. A total of 159 patients were enrolled. Overall median (interquartile range [IQR]) parasitemia half-life (half-life) was 6.03 (4.89–7.28) hours in Pailin versus 3.42 (2.20–4.85) hours in Wang Pha (P = .0001). Splitting or increasing the artesunate dose did not shorten half-life in either site. Pharmacokinetic profiles of artesunate and dihydroartemisinin were similar between sites and did not correlate with half-life. Recrudescent infections occurred in 4 of 79 patients in Pailin and 5 of 80 in Wang Pha and was not different between treatment arms (P = .68).
Conclusions. Increasing the artesunate treatment dose up to 8 mg/kg/d or splitting the dose does not improve parasite clearance in either artemisinin resistant or more sensitive infections with P. falciparum.
Clinical Trials Registration. ISRCTN15351875.
doi:10.1093/cid/cis958
PMCID: PMC3563392  PMID: 23175556
artemisinins; drug resistance; Plasmodium falciparum; neutropenia; reticulocytopenia
10.  Artemisinin Resistance in Plasmodium falciparum Malaria 
The New England journal of medicine  2009;361(5):455-467.
BACKGROUND
Artemisinin-based combination therapies are the recommended first-line treatments of falciparum malaria in all countries with endemic disease. There are recent concerns that the efficacy of such therapies has declined on the Thai–Cambodian border, historically a site of emerging antimalarial-drug resistance.
METHODS
In two open-label, randomized trials, we compared the efficacies of two treatments for uncomplicated falciparum malaria in Pailin, western Cambodia, and Wang Pha, northwestern Thailand: oral artesunate given at a dose of 2 mg per kilogram of body weight per day, for 7 days, and artesunate given at a dose of 4 mg per kilogram per day, for 3 days, followed by mefloquine at two doses totaling 25 mg per kilogram. We assessed in vitro and in vivo Plasmodium falciparum susceptibility, artesunate pharmacokinetics, and molecular markers of resistance.
RESULTS
We studied 40 patients in each of the two locations. The overall median parasite clearance times were 84 hours (interquartile range, 60 to 96) in Pailin and 48 hours (interquartile range, 36 to 66) in Wang Pha (P<0.001). Recrudescence confirmed by means of polymerase-chain-reaction assay occurred in 6 of 20 patients (30%) receiving artesunate monotherapy and 1 of 20 (5%) receiving artesunate–mefloquine therapy in Pailin, as compared with 2 of 20 (10%) and 1 of 20 (5%), respectively, in Wang Pha (P = 0.31). These markedly different parasitologic responses were not explained by differences in age, artesunate or dihydroartemisinin pharmacokinetics, results of isotopic in vitro sensitivity tests, or putative molecular correlates of P. falciparum drug resistance (mutations or amplifications of the gene encoding a multidrug resistance protein [PfMDR1] or mutations in the gene encoding sarco–endoplasmic reticulum calcium ATPase6 [PfSERCA]). Adverse events were mild and did not differ significantly between the two treatment groups.
CONCLUSIONS
P. falciparum has reduced in vivo susceptibility to artesunate in western Cambodia as compared with northwestern Thailand. Resistance is characterized by slow parasite clearance in vivo without corresponding reductions on conventional in vitro susceptibility testing. Containment measures are urgently needed. (ClinicalTrials.gov number, NCT00493363, and Current Controlled Trials number, ISRCTN64835265.)
doi:10.1056/NEJMoa0808859
PMCID: PMC3495232  PMID: 19641202
11.  In vivo susceptibility of Plasmodium falciparum to artesunate in Binh Phuoc Province, Vietnam 
Malaria Journal  2012;11:355.
Background
By 2009, there were worrying signs from western Cambodia that parasitological responses to artesunate-containing treatment regimens for uncomplicated Plasmodium falciparum malaria were slower than elsewhere which suggested the emergence of artemisinin resistance. Vietnam shares a long land border with Cambodia with a large number of migrants crossing it on a daily basis. Therefore, there is an urgent need to investigate whether there is any evidence of a change in the parasitological response to the artemisinin derivatives in Vietnam.
Methods
From August 2010 to May 2011, a randomized controlled clinical trial in uncomplicated falciparum malaria was conducted to compare two doses of artesunate (AS) (2mg/kg/day versus 4 mg/kg/day for three days) followed by dihydroartemisinin-piperaquine (DHA-PPQ) and a control arm of DHA-PPQ. The goal was characterization of the current efficacy of artesunate in southern Vietnam. The primary endpoint of this study was the parasite clearance half-life; secondary endpoints included the parasite reduction ratios at 24 and 48 hours and the parasite clearance time.
Results
166 patients were recruited into the study. The median parasite clearance half-lives were 3.54 (AS 2mg/kg), 2.72 (AS 4mg/kg), and 2.98 hours (DHA-PPQ) (p=0.19). The median parasite-reduction ratio at 24 hours was 48 in the AS 2mg/kg group compared with 212 and 113 in the other two groups, respectively (p=0.02). The proportions of patients with a parasite clearance time of >72 hours for AS 2mg/kg, AS 4mg/kg and DHA-PPQ were 27%, 27%, and 22%, respectively. Early treatment failure occurred in two (4%) and late clinical failure occurred in one (2%) of the 55 patients in the AS 2mg/kg group, as compared with none in the other two study arms. The PCR-corrected adequate clinical and parasitological response (APCR) rates in the three groups were 94%, 100%, and 100% (p=0.04).
Conclusions
This study demonstrated faster P. falciparum parasite clearance in southern Vietnam than in western Cambodia but slower clearance in comparison with historical data from Vietnam. Further studies to determine whether this represents the emergence of artemisinin resistance in this area are needed. Currently, the therapeutic response to DHA-PPQ remains satisfactory in southern Vietnam.
Trial registration
NTC01165372
doi:10.1186/1475-2875-11-355
PMCID: PMC3504531  PMID: 23101492
Plasmodium falciparum; Artesunate; Parasite clearance half-life; Parasite reduction ratio; Parasite clearance of >72 hours
12.  The global challenge of antimalarial drug resistance 
Malaria Journal  2012;11(Suppl 1):O37.
doi:10.1186/1475-2875-11-S1-O37
PMCID: PMC3472305
13.  Artemisinin-Resistant Malaria: Research Challenges, Opportunities, and Public Health Implications 
Artemisinin-based combination therapies are the most effective drugs to treat Plasmodium falciparum malaria. Reduced sensitivity to artemisinin monotherapy, coupled with the emergence of parasite resistance to all partner drugs, threaten to place millions of patients at risk of inadequate treatment of malaria. Recognizing the significance and immediacy of this possibility, the Fogarty International Center and the National Institute of Allergy and Infectious Diseases of the U.S. National Institutes of Health convened a conference in November 2010 to bring together the diverse array of stakeholders responding to the growing threat of artemisinin resistance, including scientists from malarious countries in peril. This conference encouraged and enabled experts to share their recent unpublished data from studies that may improve our understanding of artemisinin resistance. Conference sessions addressed research priorities to forestall artemisinin resistance and fostered collaborations between field- and laboratory-based researchers and international programs, with the aim of translating new scientific evidence into public health solutions. Inspired by this conference, this review summarizes novel findings and perspectives on artemisinin resistance, approaches for translating research data into relevant public health information, and opportunities for interdisciplinary collaboration to combat artemisinin resistance.
doi:10.4269/ajtmh.2012.12-0025
PMCID: PMC3414557  PMID: 22855752
14.  No Evidence for Spread of Plasmodium falciparum Artemisinin Resistance to Savannakhet Province, Southern Laos 
We conducted an open-label, randomized clinical trial to assess parasite clearance times (PCT) and the efficacy of 4 mg/kg (group 1, n = 22) and 2 mg/kg (group 2, n = 22) of oral artesunate for three days followed by artemether-lumefantrine in patients with uncomplicated Plasmodium falciparum malaria at Xepon Interdistrict Hospital, Savannakhet Province in southern Laos. Slides were read in duplicate. The overall mean (95% confidence interval; range) PCT in hours was 23.2 (21.2–25.3; 12–46) and 22.4 (20.3–24.5; 12–46) for the first and second microscopists, respectively (P = 0.57). Ten (23%) patients remained parasitemic on day 1 after treatment (4 [18%] in group 1 and 6 [27%] in group 2; P = 0.47). No patient had patent asexual parasitemia on the second and third days of treatment. The 42-day polymerase chain reaction–corrected cure rates were 100% in both treatment groups. Serious adverse events did not develop during or after treatment in any patients. In conclusion, no evidence of P. falciparum in vivo resistance to artesunate was found in southern Laos.
doi:10.4269/ajtmh.2012.11-0497
PMCID: PMC3284353  PMID: 22403308
15.  Confirmed Vivax Resistance to Chloroquine and Effectiveness of Artemether-Lumefantrine for the Treatment of Vivax Malaria in Ethiopia 
Chloroquine (CQ) is still the drug of choice for the treatment of vivax malaria in Ethiopia, whereas artemether-lumefantrine (AL) is for falciparum malaria. In this setting, clinical malaria cases are treated with AL. This necessitated the need to assess the effectiveness of AL for the treatment of Plasmodium vivax with CQ as a comparator. A total of 57 (80.3%) and 75 (85.2%) cases treated with CQ or AL, respectively, completed the study in an outpatient setting. At the end of the follow-up period of 28 days, a cumulative incidence of treatment failure of 7.5% (95% confidence interval [CI] = 2.9–18.9%) for CQ and 19% (95% CI = 11–31.6%) for AL was detected. CQ resistance was confirmed in three of five CQ treatment failures cases. The effectiveness of AL seems lower than CQ; however, the findings were not conclusive, because the AL evening doses were not supervised.
doi:10.4269/ajtmh.2011.09-0723
PMCID: PMC3005510  PMID: 21212216
16.  Decreased In Vitro Susceptibility of Plasmodium falciparum Isolates to Artesunate, Mefloquine, Chloroquine, and Quinine in Cambodia from 2001 to 2007 ▿  
This study describes the results of in vitro antimalarial susceptibility assays and molecular polymorphisms of Plasmodium falciparum isolates from Cambodia. The samples were collected from patients enrolled in therapeutic efficacy studies (TES) conducted by the Cambodian National Malaria Control Program for the routine efficacy monitoring of artemisinin-based combination therapy (ACT) (artesunate-mefloquine and artemether-lumefantrine combinations). The isolates (n = 2,041) were obtained from nine sentinel sites during the years 2001 to 2007. Among these, 1,588 were examined for their in vitro susceptibilities to four antimalarials (artesunate, mefloquine, chloroquine, and quinine), and 851 isolates were genotyped for single nucleotide polymorphisms (SNPs). The geometric means of the 50% inhibitory concentrations (GMIC50s) of the four drugs tested were significantly higher for isolates from western Cambodia than for those from eastern Cambodia. GMIC50s for isolates from participants who failed artesunate-mefloquine therapy were significantly higher than those for patients who were cured (P, <0.001). In vitro correlation of artesunate with the other drugs was observed. The distributions of the SNPs differed between eastern and western Cambodia, suggesting different genetic backgrounds of the parasite populations in these two parts of the country. The GMIC50s of the four drugs tested increased significantly in eastern Cambodia during 2006 to 2007. These results are worrisome, because they may signal deterioration of the efficacy of artesunate-mefloquine beyond the Cambodian-Thai border.
doi:10.1128/AAC.01304-09
PMCID: PMC2863643  PMID: 20194689
17.  A systematic review and meta-analysis of evidence for correlation between molecular markers of parasite resistance and treatment outcome in falciparum malaria 
Malaria Journal  2009;8:89.
Background
An assessment of the correlation between anti-malarial treatment outcome and molecular markers would improve the early detection and monitoring of drug resistance by Plasmodium falciparum. The purpose of this systematic review was to determine the risk of treatment failure associated with specific polymorphisms in the parasite genome or gene copy number.
Methods
Clinical studies of non-severe malaria reporting on target genetic markers (SNPs for pfmdr1, pfcrt, dhfr, dhps, gene copy number for pfmdr1) providing complete information on inclusion criteria, outcome, follow up and genotyping, were included. Three investigators independently extracted data from articles. Results were stratified by gene, codon, drug and duration of follow-up. For each study and aggregate data the random effect odds ratio (OR) with 95%CIs was estimated and presented as Forest plots. An OR with a lower 95th confidence interval > 1 was considered consistent with a failure being associated to a given gene mutation.
Results
92 studies were eligible among the selection from computerized search, with information on pfcrt (25/159 studies), pfmdr1 (29/236 studies), dhfr (18/373 studies), dhps (20/195 studies). The risk of therapeutic failure after chloroquine was increased by the presence of pfcrt K76T (Day 28, OR = 7.2 [95%CI: 4.5–11.5]), pfmdr1 N86Y was associated with both chloroquine (Day 28, OR = 1.8 [95%CI: 1.3–2.4]) and amodiaquine failures (OR = 5.4 [95%CI: 2.6–11.3, p < 0.001]). For sulphadoxine-pyrimethamine the dhfr single (S108N) (Day 28, OR = 3.5 [95%CI: 1.9–6.3]) and triple mutants (S108N, N51I, C59R) (Day 28, OR = 3.1 [95%CI: 2.0–4.9]) and dhfr-dhps quintuple mutants (Day 28, OR = 5.2 [95%CI: 3.2–8.8]) also increased the risk of treatment failure. Increased pfmdr1 copy number was correlated with treatment failure following mefloquine (OR = 8.6 [95%CI: 3.3–22.9]).
Conclusion
When applying the selection procedure for comparative analysis, few studies fulfilled all inclusion criteria compared to the large number of papers identified, but heterogeneity was limited. Genetic molecular markers were related to an increased risk of therapeutic failure. Guidelines are discussed and a checklist for further studies is proposed.
doi:10.1186/1475-2875-8-89
PMCID: PMC2681474  PMID: 19413906
18.  Pfmdr1 copy number and arteminisin derivatives combination therapy failure in falciparum malaria in Cambodia 
Malaria Journal  2009;8:11.
Background
The combination of artesunate and mefloquine was introduced as the national first-line treatment for Plasmodium falciparum malaria in Cambodia in 2000. However, recent clinical trials performed at the Thai-Cambodian border have pointed to the declining efficacy of both artesunate-mefloquine and artemether-lumefantrine. Since pfmdr1 modulates susceptibility to mefloquine and artemisinin derivatives, the aim of this study was to assess the link between pfmdr1 copy number, in vitro susceptibility to individual drugs and treatment failure to combination therapy.
Methods
Blood samples were collected from P. falciparum-infected patients enrolled in two in vivo efficacy studies in north-western Cambodia: 135 patients were treated with artemether-lumefantrine (AL group) in Sampovloun in 2002 and 2003, and 140 patients with artesunate-mefloquine (AM group) in Sampovloun and Veal Veng in 2003 and 2004. At enrollment, the in vitro IC50 was tested and the strains were genotyped for pfmdr1 copy number by real-time PCR.
Results
The pfmdr1 copy number was analysed for 115 isolates in the AM group, and for 109 isolates in the AL group. Parasites with increased pfmdr1 copy number had significantly reduced in vitro susceptibility to mefloquine, lumefantrine and artesunate. There was no association between pfmdr1 polymorphisms and in vitro susceptibilities. In the patients treated with AM, the mean pfmdr1copy number was lower in subjects with adequate clinical and parasitological response compared to those who experienced late treatment failure (n = 112, p < 0.001). This was not observed in the patients treated with AL (n = 96, p = 0.364). The presence of three or more copies of pfmdr1 were associated with recrudescence in artesunate-mefloquine treated patients (hazard ratio (HR) = 7.80 [95%CI: 2.09–29.10], N = 115), p = 0.002) but not with recrudescence in artemether-lumefantrine treated patients (HR = 1.03 [95%CI: 0.24–4.44], N = 109, p = 0.969).
Conclusion
This study shows that pfmdr1 copy number is a molecular marker of AM treatment failure in falciparum malaria on the Thai-Cambodian border. However, while it is associated with increased IC50 for lumefantrine, pfmdr1 copy number is not associated with AL treatment failure in the area, suggesting involvement of other molecular mechanisms in AL treatment failures in Cambodia.
doi:10.1186/1475-2875-8-11
PMCID: PMC2627910  PMID: 19138391
19.  Shelf Life of Predosed Plates Containing Mefloquine, Artemisinin, Dihydroartemisinin, and Artesunate as Used for In Vitro Plasmodium falciparum Susceptibility Assessment▿  
Journal of Clinical Microbiology  2007;45(8):2734-2736.
The shelf lives of preserved antimalarial agent-predosed plates according to the type of wrapping and the temperature of storage were studied by measuring the 50% inhibitory concentrations of drug for Plasmodium falciparum 3D7. The shelf life of mefloquine was 8 weeks at 25°C; and those of artesunate, artemisinin, and dihydroartemisinin were a minimum of 24, 12, and 8 weeks, respectively, at 4°C.
doi:10.1128/JCM.00479-07
PMCID: PMC1951262  PMID: 17553969
20.  World Antimalarial Resistance Network (WARN) II: In vitro antimalarial drug susceptibility 
Malaria Journal  2007;6:120.
Intrinsic resistance of Plasmodium falciparum is clearly a major determinant of the clinical failure of antimalarial drugs. However, complex interactions between the host, the parasite and the drug obscure the ability to define parasite drug resistance in vivo. The in vitro antimalarial drug susceptibility assay determines ex-vivo growth of parasite in the presence of serial drug concentrations and, thus, eliminates host effects, such as drug metabolism and immunity. Although the sensitivity of the parasite to various antimalarials provided by such a test provides an important indicator of intrinsic parasite susceptibility, there are fundamental methodological issues that undermine comparison of in vitro susceptibility both between laboratories and within a single laboratory over time. A network of laboratories is proposed that will agree on the basic parameters of the in vitro test and associated measures of quality control. The aim of the network would be to establish baseline values of sensitivity to commonly used antimalarial agents from key regions of the world, and create a global database, linked to clinical, molecular and pharmacology databases, to support active surveillance to monitor temporal trends in parasite susceptibility. Such a network would facilitate the rapid detection of strains with novel antimalarial resistance profiles and investigate suitable alternative treatments with retained efficacy.
doi:10.1186/1475-2875-6-120
PMCID: PMC2008206  PMID: 17822533
21.  World Antimalarial Resistance Network I: Clinical efficacy of antimalarial drugs 
Malaria Journal  2007;6:119.
The proliferation of antimalarial drug trials in the last ten years provides the opportunity to launch a concerted global surveillance effort to monitor antimalarial drug efficacy. The diversity of clinical study designs and analytical methods undermines the current ability to achieve this. The proposed World Antimalarial Resistance Network (WARN) aims to establish a comprehensive clinical database from which standardised estimates of antimalarial efficacy can be derived and monitored over time from diverse geographical and endemic regions. The emphasis of this initiative is on five key variables which define the therapeutic response. Ensuring that these data are collected at the individual patient level in a consistent format will facilitate better data management and analytical practices, and ensure that clinical data can be readily collated and made amenable for pooled analyses. Such an approach, if widely adopted will permit accurate and timely recognition of trends in drug efficacy. This will guide not only appropriate interventions to deal with established multidrug resistant strains of malaria, but also facilitate prompt action when new strains of drug resistant plasmodia first emerge. A comprehensive global database incorporating the key determinants of the clinical response with in vitro, molecular and pharmacokinetic parameters will bring together relevant data on host, drug and parasite factors that are fundamental contributors to treatment efficacy. This resource will help guide rational drug policies that optimize antimalarial drug use, in the hope that the emergence and spread of resistance to new drugs can be, if not prevented, at least delayed.
doi:10.1186/1475-2875-6-119
PMCID: PMC2008205  PMID: 17822532
22.  Comparison of a SYBR Green I-Based Assay with a Histidine-Rich Protein II Enzyme-Linked Immunosorbent Assay for In Vitro Antimalarial Drug Efficacy Testing and Application to Clinical Isolates▿  
In vitro drug susceptibility testing with the malaria parasite has been used to assess the antimalarial activities of new compounds and to monitor drug resistance in field isolates. We investigated the validity of a SYBR green I fluorescent-based assay under various culture conditions and compared the assay results to those of previously published histidine-rich protein II (HRPII) enzyme-linked immunosorbent assay (ELISA) methods. Reference strains of Plasmodium falciparum were cultured in vitro by using standard conditions in complete medium with and without phenol red before they were dispensed into 96-well plates predosed with chloroquine, mefloquine, or quinine. Following incubation, the culture supernatants were divided and the 50% inhibitory concentrations (IC50s) were determined by using a SYBR green I-based method and the HRPII capture ELISA method. There were no significant differences in IC50 values when phenol red was included in the medium. The IC50s and the IC90s of the antimalarials tested by both methods were similar or identical for each of the reference strains. Fresh clinical isolates of P. falciparum collected from imported cases of malaria in Lyon, France, were tested for in vitro resistance to chloroquine and mefloquine by using the validated SYBR green I and HRPII ELISA methods. The SYBR green I-based method was able to calculate IC50 and IC90 values similar or identical to those calculated by the HRPII assay with fresh clinical samples without removal of white blood cells. The SYBR green I-based method for determination of drug sensitivity levels produced results comparable to those produced by other methods, showing that this method can be used routinely to conduct surveillance for drug resistance in P. falciparum with fresh or cultured parasites.
doi:10.1128/AAC.01313-06
PMCID: PMC1855478  PMID: 17220424
23.  Clinical efficacy of chloroquine versus artemether-lumefantrine for Plasmodium vivax treatment in Thailand 
Chloroquine remains the drug of choice for the treatment of vivax malaria in Thailand. Mixed infections of falciparum and vivax malaria are also common in South-East Asia. Laboratory confirmation of malaria species is not generally available. This study aimed to find alternative regimens for treating both malaria species by using falciparum antimalarial drugs. From June 2004 to May 2005, 98 patients with Plasmodium vivax were randomly treated with either artemether-lumefantrine (n = 47) or chloroquine (n = 51). Both treatments were followed by 15 mg of primaquine over 14 days. Adverse events and clinical and parasitological outcomes were recorded and revealed similar in both groups. The cure rate was 97.4% for the artemether-lumefantrine treated group and 100% for the chloroquine treated group. We concluded that the combination of artemether-lumefantrine and primaquine was well tolerated, as effective as chloroquine and primaquine, and can be an alternative regimen for treatment of vivax malaria especially in the event that a mixed infection of falciparum and vivax malaria could not be ruled out.
doi:10.3347/kjp.2007.45.2.111
PMCID: PMC2526312  PMID: 17570973
Plasmodium vivax; vivax malaria; chloroquine; artemether-lumefantrine; Thailand
24.  A database of antimalarial drug resistance 
Malaria Journal  2006;5:48.
A large investment is required to develop, license and deploy a new antimalarial drug. Too often, that investment has been rapidly devalued by the selection of parasite populations resistant to the drug action. To understand the mechanisms of selection, detailed information on the patterns of drug use in a variety of environments, and the geographic and temporal patterns of resistance is needed. Currently, there is no publically-accessible central database that contains information on the levels of resistance to antimalaria drugs.
This paper outlines the resources that are available and the steps that might be taken to create a dynamic, open access database that would include current and historical data on clinical efficacy, in vitro responses and molecular markers related to drug resistance in Plasmodium falciparum and Plasmodium vivax. The goal is to include historical and current data on resistance to commonly used drugs, like chloroquine and sulfadoxine-pyrimethamine, and on the many combinations that are now being tested in different settings. The database will be accessible to all on the Web.
The information in such a database will inform optimal utilization of current drugs and sustain the longest possible therapeutic life of newly introduced drugs and combinations. The database will protect the valuable investment represented by the development and deployment of novel therapies for malaria.
doi:10.1186/1475-2875-5-48
PMCID: PMC1533841  PMID: 16774688
25.  Potent Inhibitors of Plasmodium Phospholipid Metabolism with a Broad Spectrum of In Vitro Antimalarial Activities 
We characterized the potent in vitro antimalarial activity and biologic assessment of 13 phospholipid polar head analogs on a comparative basis. There was a positive relationship between the abilities of the drugs to inhibit parasite growth in culture and their abilities to specifically inhibit phosphatidylcholine biosynthesis of Plasmodium falciparum-infected erythrocytes. Maximal activity of G25 was observed for the trophozoite stage of the 48-h erythrocytic cycle (50% inhibitory concentration, 0.75 nM), whereas the schizont and ring stages were 12- and 213-fold less susceptible. The compounds exerted a rapid nonreversible cytotoxic effect, with complete clearance of parasitemia after 5 h of contact with the mature stages. The compounds were highly specific against P. falciparum, with much lower toxicity against three other mammalian cell lines, and the in vitro therapeutic indices ranged from 300 to 2,500,000. Finally, the monoquaternary ammonium E10 and two bis-ammonium salts, G5 and G25, were similarly active against multiresistant strains and fresh isolates of P. falciparum. This impressive selective in vitro toxicity against P. falciparum strongly highlights the clinical potential of these quaternary ammonium salts for malarial chemotherapy.
doi:10.1128/AAC.47.8.2590-2597.2003
PMCID: PMC166094  PMID: 12878524

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