Pharmacokinetics and pharmacodynamics of oral artesunate monotherapy in patients with uncomplicated Plasmodium falciparum malaria in western Cambodia.

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.

Ex vivo drug sensitivity profiles of Plasmodium falciparum field isolates from Cambodia and Thailand, 2005 to 2010, determined by a histidine-rich protein-2 assay.

BACKGROUND: In vitro drug susceptibility assay of Plasmodium falciparum field isolates processed "immediate ex vivo" (IEV), without culture adaption, and tested using histidine-rich protein-2 (HRP-2) detection as an assay, is an expedient way to track drug resistance. METHODS: From 2005 to 2010, a HRP-2 in vitro assay assessed 451 P. falciparum field isolates obtained from subjects with malaria in western and northern Cambodia, and eastern Thailand, processed IEV, for 50% inhibitory concentrations (IC50) against seven anti-malarial drugs, including artesunate (AS), dihydroartemisinin (DHA), and piperaquine. RESULTS: In western Cambodia, from 2006 to 2010, geometric mean (GM) IC50 values for chloroquine, mefloquine, quinine, AS, DHA, and lumefantrine increased. In northern Cambodia, from 2009-2010, GM IC50 values for most drugs approximated the highest western Cambodia GM IC50 values in 2009 or 2010. CONCLUSIONS: Western Cambodia is associated with sustained reductions in anti-malarial drug susceptibility, including the artemisinins, with possible emergence, or spread, to northern Cambodia. This potential public health crisis supports continued in vitro drug IC50 monitoring of P. falciparum isolates at key locations in the region.

Towards high-throughput molecular detection of Plasmodium: new approaches and molecular markers.

BACKGROUND: Several strategies are currently deployed in many countries in the tropics to strengthen malaria control toward malaria elimination. To measure the impact of any intervention, there is a need to detect malaria properly. Mostly, decisions still rely on microscopy diagnosis. But sensitive diagnosis tools enabling to deal with a large number of samples are needed. The molecular detection approach offers a much higher sensitivity, and the flexibility to be automated and upgraded. METHODS: Two new molecular methods were developed: dot18S, a Plasmodium-specific nested PCR based on the 18S rRNA gene followed by dot-blot detection of species by using species-specific probes and CYTB, a Plasmodium-specific nested PCR based on cytochrome b gene followed by species detection using SNP analysis. The results were compared to those obtained with microscopic examination and the "standard" 18S rRNA gene based nested PCR using species specific primers. 337 samples were diagnosed. RESULTS: Compared to the microscopy the three molecular methods were more sensitive, greatly increasing the estimated prevalence of Plasmodium infection, including P. malariae and P. ovale. A high rate of mixed infections was uncovered with about one third of the villagers infected with more than one malaria parasite species. Dot18S and CYTB sensitivity outranged the "standard" nested PCR method, CYTB being the most sensitive. As a consequence, compared to the "standard" nested PCR method for the detection of Plasmodium spp., the sensitivity of dot18S and CYTB was respectively 95.3% and 97.3%. Consistent detection of Plasmodium spp. by the three molecular methods was obtained for 83% of tested isolates. Contradictory results were mostly related to detection of Plasmodium malariae and Plasmodium ovale in mixed infections, due to an "all-or-none" detection effect at low-level parasitaemia. CONCLUSION: A large reservoir of asymptomatic infections was uncovered using the molecular methods. Dot18S and CYTB, the new methods reported herein are highly sensitive, allow parasite DNA extraction as well as genus- and species-specific diagnosis of several hundreds of samples, and are amenable to high-throughput scaling up for larger sample sizes. Such methods provide novel information on malaria prevalence and epidemiology and are suited for active malaria detection. The usefulness of such sensitive malaria diagnosis tools, especially in low endemic areas where eradication plans are now on-going, is discussed in this paper.

Access to artemisinin combination therapy for malaria in remote areas of Cambodia.

BACKGROUND: Malaria-endemic countries are switching antimalarial drug policy to artemisinin combination therapies (ACTs) and the global community are considering the setting up of a global subsidy mechanism in order to make them accessible and affordable. However, specific interventions may be needed to reach remote at-risk communities and to ensure that they are used appropriately. This analysis documents the coverage with ACTs versus artemisinin monotherapies, and the effectiveness of malaria outreach teams (MOTs) and Village Malaria Workers (VMWs) in increasing access to appropriate diagnosis and treatment with ACTs in Cambodia, the first country to switch national antimalarial drug policy to an ACT of artesunate and mefloquine (A+M) in 2000. METHODS: A cross-sectional survey was carried out in three different types of intervention area: with VMWs, MOTs and no specific interventions. Individuals with a history of fever in the last three weeks were included in the study and completed a questionnaire on their treatment seeking and drug usage behaviour. Blood was taken for a rapid diagnostic test (RDT) and data on the household socio-economic status were also obtained. RESULTS: In areas without specific interventions, only 17% (42/251) of respondents received a biological diagnosis, 8% (17/206) of respondents who received modern drug did so from a public health facility, and only 8% of them (17/210) received A+M. Worryingly, 78% (102/131) of all artemisinin use in these areas was as a monotherapy. However, both the VMW scheme and MOT scheme significantly increased the likelihood of being seen by a trained provider (Adjusted Odds Ratios (AOR) of 148 and 4 respectively) and of receiving A+M (AORs of 2.7 and 7.7 respectively). CONCLUSION: The coverage rates of appropriate diagnosis and treatment of malaria were disappointingly low and the use of artemisinin monotherapy alarmingly high. This reflects the fragmented nature of Cambodia's health system in remote areas and the reliance placed by these communities on informal vendors from whom artemisinin monotherapies are widely available. However VMWs in particular are an effective means of improving access to malaria diagnosis and treatment. The VMW scheme and the social marketing of RDTS and blister-packaged artesunate and mefloquine have both been scaled up nationally. Case management in the public sector has also reportedly improved. Given recent concerns regarding the development of artemisinin drug resistance on the Thai-Cambodia border, the effectiveness of these measures in reducing the use of artemisinin monotherapy needs to be urgently re-evaluated.

Changing patterns of forest malaria among the mobile adult male population in Chumkiri District, Cambodia.

Forest malaria remains a major problem in many parts of Southeast Asia and South America. In Cambodia, where a significant reduction of malaria morbidity and mortality has been observed in the last 20 years, the forest malaria situation was studied in Chumkiri District by analysing the available passive case detection data and conducting malariometric (n=1018) and questionnaire surveys (n=374) in four forest-fringe villages. There has been a decreasing trend of malaria incidence from 2001. Plasmodium falciparum was highly predominant and P. vivax was rare. The nearby-forest villages showed significantly higher parasite rates than the far-from-forest villages (9.0% vs. 1.2%, p<0.01). Malaria was highly restricted to the male adults but was nearly non-existent in other accompanying family members, including small children and females. Low income and working in forests were strongly associated with the malaria risk. Our results suggest that transmission has greatly reduced in forest-fringe villages, but remains active in forests, which is primarily maintained between the forest vector Anopheles dirus and ethnic minority inhabitants. Specific interventions directed to these previously neglected in-forest inhabitants to protect themselves and male adult villagers during their forest activities are necessary to achieve an ultimate goal of malaria elimination from Cambodia.

The effect of long-lasting insecticidal water container covers on field populations of Aedes aegypti (L.) mosquitoes in Cambodia.

Dengue in Cambodia is mainly transmitted by Aedes aegypti (L.) mosquitoes that primarily breed in large, concrete jars (> or =200 liters) used for the storage of water for domestic use. Following a preliminary risk assessment, long-lasting insecticidal netting (LN) treated with deltamethrin was incorporated into the design of the covers for these jars. Their effect on immature and adult female populations of Ae. aegypti in six villages in a peri-urban area of Cambodia were compared with populations in six nearby control villages before and for 22 weeks after distribution of the jar covers. There were significantly fewer pupae per house in intervention villages than in control villages (6.6 and 31.9, respectively, p<0.01). Fewer pupae were recovered from intervention houses than from control houses at every post-intervention assessment. Two weeks after the intervention, the average number of indoor resting female Ae. aegypti per house in the intervention villages had declined approximately three-fold, whereas in the controls there was only a slight reduction (16%). The magnitude of the difference between the two areas diminished over time, which contact bioassays confirmed was likely due to a gradual reduction of insecticidal effect of the jar covers. In the study area, insecticide-treated covers for large concrete water storage jars were efficacious for controlling Ae. aegypti in the protected water jars and with a demonstrable effect on adult densities and survival. Further studies of this targeted container strategy in Cambodia, and elsewhere, are recommended. However, improvements in technology that would extend the duration of insecticidal effectiveness of LN materials may be needed for the development of cost-effective public health applications.

Community-based use of the larvivorous fish Poecilia reticulata to control the dengue vector Aedes aegypti in domestic water storage containers in rural Cambodia.

A community-based study of the distribution of larvivorous fish, Poecilia reticulata (common name: guppy), in water storage containers for dengue control was undertaken in 14 villages and approximately 1,000 households in Cambodia. Community volunteers reared guppies and distributed them in water jars and tanks in households for which they were responsible. A nearby control area received no intervention. One year after project commencement, 56.9% of eligible containers contained guppies and there was a 79.0% reduction in Aedes infestation in the intervention community compared with the control. Smaller or discarded containers unsuitable for guppy distribution in the intervention area also had 51% less infestation than those in the control area, suggesting a "community-wide" protective effect. In addition, there was less infestation in villages with higher rates of fish uptake, suggesting that the presence of fish was responsible for a reduction in Aedes infestation. This applied vector control model was well-accepted, effective, efficient, and shows promise as a sustainable community-based, non-insecticidal intervention for dengue vector control in large domestic water storage containers in rural Cambodia and elsewhere.

Efficacy of Bacillus thuringiensis israelensis, VectoBac WG and DT, formulations against dengue mosquito vectors in cement potable water jars in Cambodia.

This study reports the evaluation of Bacillus thuringiensis israelensis (Bti), a biological larvicide, in cement jars holding river, well and rain water. Two Bti formulations, VectoBac WG and VectoBac DT, were evaluated in a village in Phnom Penh. Thirty-one households with cement jars supporting the colonization of Ae.aegypti immatures were chosen. In each house 3 jars were aligned next to each another and filled with the same type of water. One of the 3 jars was treated with VectoBac WG at 0.4 g per 50 liters, a second jar was treated with VectoBac DT at 1 tablet per 50 liters, and a third jar was an untreated control (UTC). The jars were not covered, kept outdoors and not subjected to water exchange activity. The efficacy of VectoBac to control natural Ae.aegypti infestation was measured by Ae.aegypti pupae surveillance, conducted 3 days per week for 3 months post-treatment (June-September 2004). All pupae were removed, allowed to emerge in the Cambodia National Malaria Center insectarium and the emerged adults were identified and counted. The VectoBac treatments were more effective in river water, followed by well and rain water. The VectoBac treatments significantly reduced the pupae numbers for a minimum of 3 months in the river water and 2.5 months in the well water (p < 0.05). In the rain water, the pupae densities in the VectoBac WG and DT treated jars were not significantly different from the untreated jars, although the treated jars yielded 2.0 to 5.2 fold less pupae, respectively, than in the untreated jars during the 3 months post-treatment. The efficacy of VectoBac WG to control Ae.aegypti was similar to the efficacy of VectoBac DT in the 3 water types (p > 0.05). It was also observed that VectoBac WG and DT were target specific, without any adverse effects on aquatic predatory insects common in well and rain water. VectoBac WG and DT were found to be easy-to-use formulations, with no need to repackage them prior to use in the containers. The amounts of VectoBac WG and DT used were 12.5 fold less by weight than temephos (Abate 1.0% SG).

Bacterial Larvicide, Bacillus thuringiensis israelensis Strain AM 65-52 Water Dispersible Granule Formulation Impacts Both Dengue Vector, Aedes aegypti (L.) Population Density and Disease Transmission in Cambodia.

A multi-phased study was conducted in Cambodia from 2005-2011 to measure the impact of larviciding with the bacterial larvicide, Bacillus thuringiensis israelensis (Bti), a water dispersible granule (WG) formulation on the vector, Aedes aegypti (L.) and the epidemiology. In our studies, all in-use containers were treated at 8 g/1000 L, including smaller containers and animal feeders which were found to contribute 23% of Ae aegypti pupae. The treated waters were subjected to routine water exchange activities. Pupal production was suppressed by an average 91% for 8 weeks. Pupal numbers continued to remain significantly lower than the untreated commune (UTC) for 13 weeks post treatment in the peak dengue vector season (p<0.05). Suppression of pupal production was supported by very low adult numbers in the treated commune. An average 70% of the household harbored 0-5 Ae aegypti mosquitoes per home for 8 weeks post treatment, but in the same period of time >50% of the household in the UTC harbored ≥11 mosquitoes per home. The adult population continued to remain at significantly much lower numbers in the Bti treated commune than in the UTC for 10-12 weeks post treatment (p<0.05). In 2011, a pilot operational program was evaluated in Kandal Province, a temephos resistant site. It was concluded that 2 cycles of Bti treatment in the 6 months monsoon season with complete coverage of the target districts achieved an overall dengue case reduction of 48% in the 6 treated districts compared to the previous year, 2010. Five untreated districts in the same province had an overwhelming increase of 352% of dengue cases during the same period of time. The larvicide efficacy, treatment of all in-use containers at the start of the monsoon season, together with treatment coverage of entire districts interrupted disease transmission in the temephos resistant province.

Financial costs of deworming children in all primary schools in Cambodia.

In 2002, Cambodia's Ministry of Health launched a deworming programme to deliver an anthelmintic drug (mebendazole 500 mg) and health education to 75% of its school children twice a year. Cambodia's school population is approximately 2.8 million. The deworming programme was organized into two phases: the first phase (December 2002-March 2003) targeted more than one million school children from 11 provinces; and the second phase (July 2003-January 2004) targeted the entire school population. The cost to treat each child was 12 cents (0.11 USD) during the first phase, 6 cents during the second phase, and 3 cents for re-treatment in areas where the campaign was conducted for the second time. The Cambodian experience demonstrates that, with political commitment, high coverage for deworming is achievable even in a country with minimal resources. Cambodia's deworming programme represents a successful model for other developing countries.

In vitro monitoring of Plasmodium falciparum susceptibility to artesunate, mefloquine, quinine and chloroquine in Cambodia: 2001-2002.

We used a classical isotopic microtest to assess the in vitro sensitivity of 352 Plasmodium falciparum isolates collected in Cambodia in 2001 and 2002 to chloroquine, mefloquine, quinine and artesunate. Our results confirm conclusions drawn from earlier studies conducted by the Cambodian national malaria centre. Chloroquine-resistant phenotypes were highly prevalent in Cambodia. Similarly, a high proportion of isolates displayed elevated IC50 to mefloquine. In contrast, only 0.67 and 1.7% of isolates presented decreased susceptibility to quinine and artesunate, respectively. Distributions of mean IC50 according to drug and geographic origin indicated that the parasites circulating to the west of Cambodia largely account for the global situation of drug resistances in Cambodia. Isolates with decreased susceptibility to chloroquine and mefloquine were common along the border with Thailand. In contrast, most of the isolates from eastern Cambodia were susceptible to these compounds. Isolates collected at the western and eastern borders did not respond differently to artesunate. No major differences in responses to antimalarial drugs were observed between 2001 and 2002, suggesting that the situation of drug resistance is now stabilized and under control in Cambodia. However, the decreased susceptibility of isolates collected in the western provinces of Cambodia to mefloquine and the correlation between susceptibility to artesunate and susceptibility to mefloquine and quinine justify the need for an improved international surveillance program for malaria drug resistance in the Mekong sub region.

Efficacy and safety of dihydroartemisinin-piperaquine (Artekin) in Cambodian children and adults with uncomplicated falciparum malaria.

The safety and efficacy of a novel combination of dihydroartemisinin (DHA) and piperaquine, Artekin (Holleykin Pharmaceuticals), were assessed in 106 patients (76 children and 30 adults) with uncomplicated falciparum malaria from 2 remote areas in Cambodia. Age-based doses were given at 0, 8, 24, and 32 h. Mean total DHA and piperaquine doses were 9.1 and 73.9 mg/kg, respectively, for children and 6.6 and 52.9 mg/kg for adults. All patients became aparasitemic within 72 h. Excluding the results for 1 child who died on day 4, there was a 96.9% 28-day cure rate (98.6% in children and 92.3% in adults). Patients who had recrudescent infection received low doses of Artekin. Side effects were reported by 22 patients (21%) but did not necessitate premature cessation of therapy. Although Artekin is a promising and inexpensive option for antimalarial therapy, further efficacy and pharmacokinetic studies are needed, especially for its use in children.

Intestinal helminthic infections in schoolchildren in Cambodia.

During the period January to December 1998, the National Malaria Center (CNM) carried out a parasitological survey of schoolchildren in rural and semi-urban areas, to assess intestinal helminthic infections in schoolchildren in the central parts of Cambodia. In the rural areas, there were four schools in Stung Treng Province (all situated along the Mekong River), five schools in Kratie Province (around rubber plantations), six schools in Kampong Chhnang Province (along Tonle Sap Lake); and in the semi-urban areas, three schools in Beng Tumpon Commune and five schools in Chbar Ampeou Commune (Mean Chey District) were selected for study. By Kato-Katz technique, the prevalence of soil-transmitted helminthic infections in schoolchildren in both the rural and urban areas was high. The infection rate was between 10-40% for Ascaris, 2-17% for Trichuris and 5-65% for hookworm. Schistosomiasis and opisthorchiasis were found in the schoolchildren living along the Mekong River (Stung Treng Province); the infection rate of S. mekongi ranged from 12 to 43%. These infections in children were with hepatomegalies. An intervention in an urban area (Chraing Chamres) showed that after repeated treatment with mebendazole 500 mg single dose every 6 months, the prevalence of all parasites had dropped to about one third of the initial level.

Mekong malaria. II. Update of malaria, multi-drug resistance and economic development in the Mekong region of Southeast Asia.

In an expansion of the first Mekong Malaria monograph published in 1999, this second monograph updates the malaria database in the countries comprising the Mekong region of Southeast Asia. The update adds another 3 years' information to cover cumulative data from the 6 Mekong countries (Cambodia, China/Yunnan, Lao PDR, Myanmar, Thailand, Viet Nam) for the six-year period 1999-2001. The objective is to generate a more comprehensive regional perspective in what is a global epicenter of drug resistant falciparum malaria, in order to improve malaria control on a regional basis in the context of social and economic change. The further application of geographical information systems (GIS) to the analysis has underscored the overall asymmetry of disease patterns in the region, with increased emphasis on population mobility in disease spread. Of great importance is the continuing expansion of resistance of P. falciparum to antimalarial drugs in common use and the increasing employment of differing drug combinations as a result. The variation in drug policy among the 6 countries still represents a major obstacle to the institution of region-wide restrictions on drug misuse. An important step forward has been the establishment of 36 sentinel sites throughout the 6 countries, with the objective of standardizing the drug monitoring process; while not all sentinel sites are fully operational yet, the initial implementation has already given encouraging results in relation to disease monitoring. Some decreases in malaria mortality have been recorded. The disease patterns delineated by GIS are particularly instructive when focused on inter-country distribution, which is where more local collaborative effort can be made to rationalize resource utilization and policy development. Placing disease data in the context of socio-economic trends within and between countries serves to further identify the needs and the potential for placing emphasis on resource rationalization on a regional basis. Despite the difficulties, the 6-year time frame represented in this monograph gives confidence that the now well established collaboration is becoming a major factor in improving malaria control on a regional basis and hopefully redressing to a substantial degree the key problem of spread of drug resistance regionally and eventually globally.

Safety evaluation of fixed combination piperaquine plus dihydroartemisinin (Artekin) in Cambodian children and adults with malaria.

AIMS: To assess the haemodynamic, electrocardiographic and glycaemic effects of piperaquine-dihydroartemisinin (Artekin) fixed combination therapy in uncomplicated malaria. METHODS: Sixty-two Cambodians (32 children and 30 adults) with falciparum or vivax malaria were given Artekin given as four age-based oral doses over 32 h. Supine and erect blood pressure, the electrocardiographic QT interval and plasma glucose were measured before treatment and then at regular intervals during a 4-day admission period as part of efficacy and safety monitoring. QT intervals were rate-corrected (QTc) using Bazett's formula. RESULTS: Artekin therapy was well tolerated and all patients responded to treatment. Average parasite and fever clearance times were 19 and 12 h, respectively. The pretreatment mean fall in systolic blood pressure on standing was 8 +/- 6 mmHg and 6-hourly measurements over 72 h showed no significant change (P = 0.48). There was a significant lengthening of the mean QTc to a maximum of 11 ms(0.5) (95% confidence interval 4-18 ms(0.5)) relative to baseline at 24 h (P = 0.003). The maximal QTc prolongation observed in any patient was 53 ms(0.5). There was a mean 0.4 mmol l(-1) reduction in the post-absorptive plasma glucose during the first 48 h but no episodes of hypoglycaemia (plasma glucose < 3.0 mmol l(-1)) were observed at any time. CONCLUSIONS: Artekin is safe and effective combination therapy for uncomplicated malaria in children and adults. Although piperaquine is a long half-life drug related to other quinoline compounds including chloroquine and quinine, no clinically significant cardiovascular or metabolic effects were observed.

Population pharmacokinetics of piperaquine in adults and children with uncomplicated falciparum or vivax malaria.

AIMS: To study the population pharmacokinetics of piperaquine after co-administration with dihydroartemisinin in uncomplicated malaria. METHODS: The disposition of piperaquine was studied in 85 Cambodian patients with uncomplicated falciparum or vivax malaria treated with the piperaquine-dihydroartemisinin coformulation Artekin. All patients were given Artekin orally at 0, 6, 24 and 32 h with a total piperaquine dose of 32-35 mg base kg-1. Adults were given tablets while children received either tablets or a dispersible granule formulation. Patients underwent either intensive (17-19 samples) or sparse (2-5 samples) blood sampling schedules over 35 days and clinical/parasitological follow-up over > 28 days. Piperaquine in plasma was quantified by high performance liquid chromatography. RESULTS: All patients achieved fever clearance within 24 h and parasite clearance within 72 h. The 28-day cure rate was 97% in adults and 98% in children. A covariate-free two-compartment population model with first-order absorption and elimination gave the most robust representation of the plasma concentration-time data in both adults and children. In adults (n = 38), the median (interquartile range) derived pharmacokinetic descriptors CL/F, Vss/F and t1/2,z were 0.9 l h-1 kg-1 (0.79-1.02 l h-1 kg-1), 574 l kg-1(371-711 l kg-1) and 23 days (19-28 days), respectively. In children (n = 47), corresponding values were 1.8 l h-1 kg-1 (1.29-2.3 l h-1 kg-1), 614 l kg-1 (332-1205 l kg-1) and 14 days (10-18 days), respectively. CONCLUSIONS: Piperaquine is a highly lipid-soluble drug with a large Vss/F, long t1/2,z and a clearance that is markedly higher in children than in adults.

pfcrt polymorphism and chloroquine resistance in Plasmodium falciparum strains isolated in Cambodia.

Plasmodium falciparum chloroquine resistance was first detected in Cambodia in the early sixties. Treatment with chloroquine was abandoned 20 years ago. In vitro chloroquine sensitivity monitoring indicates that all eastern Cambodian isolates were sensitive to chloroquine, whereas most isolates collected from western provinces displayed reduced susceptibility to chloroquine. This indicates that the rate of chloroquine resistance remains high and stable in this region in the absence of chloroquine pressure. Characterization of codons 72 to 78 and 218 to 220 of pfcrt revealed six distinct haplotypes, four of which had never been described. The frequency of each haplotype depended on the geographical origin of the samples. The CVIETIF//ISS haplotype was detected in 92% of western Cambodian isolates and in 11% of isolates collected from the eastern province, where CVMNKIF//ISA and CVIDTIF//ISS predominate. The detection of an intermediate haplotype from a susceptible area with 76T/220A, suggests that acquisition of chloroquine resistance might be a stepwise process, during which accumulation of point mutations modulates the response to chloroquine. The association of the K76T mutation with chloroquine resistance was not clear. The mutation was detected in resistant and susceptible samples, suggesting that additional factors are involved in chloroquine resistance. By contrast, the pfcrt D/N75E mutation was strongly associated with the in vitro chloroquine resistance in Cambodian isolates. The N86 allelic form of pfmdr1 was detected in all isolates, consistent with a poor association with resistance to chloroquine. This indicates that in vitro resistance to chloroquine was associated with accumulation of point mutations in pfcrt.