Pfcrt genotypes and related microsatellite DNA polymorphisms on Plasmodium falciparum differed among populations in the Greater Mekong Subregion.

Malaria morbidity and mortality have decreased gradually in the Greater Mekong Subregion (GMS). Presently, WHO sets a goal to eliminate malaria by 2030 in the GMS. However, drug-resistant malaria has been reported from several endemic areas. To achieve the goal of elimination, the status of the emergence and spread of drug resistance should be monitored. In this study, the genotype of the Plasmodium falciparum chloroquine (CQ) resistance transporter gene (pfcrt) and 6 microsatellite DNA loci flanking the gene were examined. P. falciparum isolates (n = 136) was collected from malaria patients in Thailand (n = 50, 2002-2005), Vietnam (n = 39, 2004), Laos (n = 15, 2007) and Cambodia (n = 32, 2009). Amino acid sequences at codons 72-76 on the gene were determined. All of the isolates from Thailand were CQ-resistant (CVIET), as were all of the isolates from Cambodia (CVIET, CVIDT). Thirteen of the 15 isolates (87%) from Laos were CQ-resistant (CVIET, CVIDT), whereas the other 2 (13%) were CQ-susceptible (CVMNK). In contrast, 27 of the 39 isolates (69%) from Vietnam were CQ-susceptible (CVMNK), whereas the other 12 (31%) were CQ-resistant (CVIET, CVIDT, CVMDT) or mixed (CVMNK/CVIDT). The mean of expected heterozygosity of the microsatellite loci was 0.444 in the Thai population, 0.482 in the Cambodian population, and 0.734 in the Vietnamese population. Genetic diversity in the Thai population was significantly lower than that in the Vietnamese population. These results suggested that chloroquine selective pressure on P. falciparum populations is heterogeneous in the GMS. Therefore, further examination to understand the mechanisms behind the emergence and spread of drug-resistant malaria are needed.

Stellantchasmus falcatus (Digenea: Heterophyidae) in Cambodia: Discovery of Metacercariae in Mullets and Recovery of Adult Flukes in an Experimental Hamster.

Stellantchasmus falcatus (Digenea: Heterophyidae) is first reported from Cambodia through recovery of the metacercariae from mullet fish and adult flukes from an experimentally infected hamster. We purchased 7 mullets, Chelon macrolepis, in a local market of Phnom Penh, Cambodia, and each of them was examined by the artificial digestion method on May 2010. The metacercariae of S. falcatus were detected in all mullets (100%) examined, and their average density was 177 per fish. They were elliptical, 220×168 µm in average size. They were orally infected to an hamster to obtain adult flukes. Adults recovered at day 10 post infection were observed with a light microscope and a scanning electron microscope (SEM). They were small, 450×237 µm in average size, had a small oral sucker (41×50 µm), subglobular pharynx (29×21 µm), slender esophagus (57 µm), long and thick-walled expulsor (119×32 µm), spherical ovary (58×69 µm), and 2 ovoid testes (right: 117×74 µm; left: 114×63 µm). Eggs were small, yellow, and 23×12 µm in average size. In SEM observations, tegumental spines were densely distributed on the whole tegument, and single small type I sensory papillae were distributed around the lip of oral sucker. The small ventral sucker was dextrally located and had 8 type I sensory papillae on the left margin. It has been first confirmed in the present study that the mullet, C. macrolepis, is playing the role of a second intermediate host of S. falcatus in Cambodia.

National Malaria Prevalence in Cambodia: Microscopy Versus Polymerase Chain Reaction Estimates.

Accurate information regarding malaria prevalence at national level is required to design and assess malaria control/elimination efforts. Although many comparisons of microscopy and polymerase chain reaction (PCR)-based methods have been conducted, there is little published literature covering such comparisons in southeast Asia especially at the national level. Both microscopic examination and PCR detection were performed on blood films and dried blood spots samples collected from 8,067 individuals enrolled in a nationwide, stratified, multistage, cluster sampling malaria prevalence survey conducted in Cambodia in 2007. The overall malaria prevalence and prevalence rates of Plasmodium falciparum, Plasmodium vivax, and Plasmodium malariae infections estimated by microscopy (N = 8,067) were 2.74% (95% confidence interval [CI]: 2.39-3.12%), 1.81% (95% CI: 1.53-2.13%), 1.14% (95% CI: 0.92-1.40%), and 0.01% (95% CI: 0.003-0.07%), respectively. The overall malaria prevalence based on PCR detection (N = 7,718) was almost 2.5-fold higher (6.31%, 95% CI: 5.76-6.89%, P < 0.00001). This difference was significantly more pronounced for P. falciparum (4.40%, 95% CI: 3.95-4.90%, P < 0.00001) compared with P. vivax (1.89%, 95% CI: 1.60-2.22%, P < 0.001) and P. malariae infections (0.22%, 95% CI: 0.13-0.35%, P < 0.0001). The significant proportion of microscopy-negative but PCR-positive individuals (289/7,491, 3.85%) suggest microscopic examination frequently underestimated malaria infections and that active case detection based on microscopy may miss a significant reservoir of infection, especially in low-transmission settings.

Prevalence of intestinal helminths among inhabitants of Cambodia (2006-2011).

In order to investigate the status of intestinal helminthic infections in Cambodia, epidemiological surveys were carried out on a national scale, including 19 provinces. A total of 32,201 fecal samples were collected from schoolchildren and adults between 2006 and 2011 and examined once by the Kato-Katz thick smear technique. The overall egg positive rate of intestinal helminths was 26.2%. The prevalence of hookworms was the highest (9.6%), followed by that of Opisthorchis viverrini/minute intestinal flukes (Ov/MIF) (5.7%), Ascaris lumbricoides (4.6%), and Trichuris trichiura (4.1%). Other types of parasites detected were Enterobius vermicularis (1.1%), Taenia spp. (0.4%), and Hymenolepis spp. (0.2%). The northwestern regions such as the Siem Reap, Oddar Meanchey, and Banteay Meanchey Provinces showed higher prevalences (17.4-22.3%) of hookworms than the other localities. The southwestern areas, including Koh Kong and Preah Sihanouk Provinces showed higher prevalences of A. lumbricoides (17.5-19.2%) and T. trichiura (6.1-21.0%). Meanwhile, the central and southern areas, in particular, Takeo and Kampong Cham Provinces, showed high prevalences of Ov/MIF (23.8-24.0%). The results indicate that a considerably high prevalence of intestinal helminths has been revealed in Cambodia, and thus sustained national parasite control projects are necessary to reduce morbidity due to parasitic infections in Cambodia.

Differing patterns of selection and geospatial genetic diversity within two leading Plasmodium vivax candidate vaccine antigens.

Although Plasmodium vivax is a leading cause of malaria around the world, only a handful of vivax antigens are being studied for vaccine development. Here, we investigated genetic signatures of selection and geospatial genetic diversity of two leading vivax vaccine antigens--Plasmodium vivax merozoite surface protein 1 (pvmsp-1) and Plasmodium vivax circumsporozoite protein (pvcsp). Using scalable next-generation sequencing, we deep-sequenced amplicons of the 42 kDa region of pvmsp-1 (n = 44) and the complete gene of pvcsp (n = 47) from Cambodian isolates. These sequences were then compared with global parasite populations obtained from GenBank. Using a combination of statistical and phylogenetic methods to assess for selection and population structure, we found strong evidence of balancing selection in the 42 kDa region of pvmsp-1, which varied significantly over the length of the gene, consistent with immune-mediated selection. In pvcsp, the highly variable central repeat region also showed patterns consistent with immune selection, which were lacking outside the repeat. The patterns of selection seen in both genes differed from their P. falciparum orthologs. In addition, we found that, similar to merozoite antigens from P. falciparum malaria, genetic diversity of pvmsp-1 sequences showed no geographic clustering, while the non-merozoite antigen, pvcsp, showed strong geographic clustering. These findings suggest that while immune selection may act on both vivax vaccine candidate antigens, the geographic distribution of genetic variability differs greatly between these two genes. The selective forces driving this diversification could lead to antigen escape and vaccine failure. Better understanding the geographic distribution of genetic variability in vaccine candidate antigens will be key to designing and implementing efficacious vaccines.

Zoonotic trematode metacercariae in fish from Phnom Penh and Pursat, Cambodia.

A survey was performed to investigate the infection status of freshwater fish with zoonotic trematode metacercariae in Phnom Penh and Pursat Province, Cambodia. All collected fish with ice were transferred to our laboratory and examined using the artificial digestion method. In fish from Phnom Penh, 2 kinds of metacercariae (Opisthorchis viverrini and Haplorchis yokogawai) were detected. O. viverrini metacercariae were positive in 37 (50.0%) of 74 fish in 11 species (average no. metacercariae/fish, 18.6). H. yokogawai metacercariae were detected in 23 (57.5%) of 40 fish in 5 species (average no. metacercariae/fish, 21.0). In fish from Pursat Province, 5 kinds of metacercariae (O. viverrini, H. yokogawai, Haplorchis pumilio, Centrocestus formosanus, and Procerovum sp.) were detected; O. viverrini metacercariae (n=3) in 2 fish species (Henicorhynchus lineatus and Puntioplites falcifer), H. yokogawai metacercariae (n=51) in 1 species (P. falcifer), H. pumilio metacercariae (n=476) in 2 species (H. lineatus and Pristolepis fasciata), C. formosanus metacercariae (n=1) in 1 species (H. lineatus), and Procerovum sp. metacercariae (n=63) in 1 species (Anabas testudineus). From the above results, it has been confirmed that various freshwater fish play the role of a second intermediate host for zoonotic trematodes (O. viverrini, H. yokogawai, H. pumilio, C. formosanus, and Procerovum sp.) in Cambodia.

Laboratory detection of artemisinin-resistant Plasmodium falciparum.

Conventional 48-h in vitro susceptibility tests have low sensitivity in identifying artemisinin-resistant Plasmodium falciparum, defined phenotypically by low in vivo parasite clearance rates. We hypothesized originally that this discrepancy was explained by a loss of ring-stage susceptibility and so developed a simple field-adapted 24-h trophozoite maturation inhibition (TMI) assay focusing on the ring stage and compared it to the standard 48-h schizont maturation inhibition (WHO) test. In Pailin, western Cambodia, where artemisinin-resistant P. falciparum is prevalent, the TMI test mean (95% confidence interval) 50% inhibitory concentration (IC50) for artesunate was 6.8 (5.2 to 8.3) ng/ml compared with 1.5 (1.2 to 1.8) ng/ml for the standard 48-h WHO test (P = 0.001). TMI IC50s correlated significantly with the in vivo responses to artesunate (parasite clearance time [r = 0.44, P = 0.001] and parasite clearance half-life [r = 0.46, P = 0.001]), whereas the standard 48-h test values did not. On continuous culture of two resistant isolates, the artemisinin-resistant phenotype was lost after 6 weeks (IC50s fell from 10 and 12 ng/ml to 2.7 and 3 ng/ml, respectively). Slow parasite clearance in falciparum malaria in western Cambodia results from reduced ring-stage susceptibility.

Optimal sampling designs for estimation of Plasmodium falciparum clearance rates in patients treated with artemisinin derivatives.

BACKGROUND: The emergence of Plasmodium falciparum resistance to artemisinins in Southeast Asia threatens the control of malaria worldwide. The pharmacodynamic hallmark of artemisinin derivatives is rapid parasite clearance (a short parasite half-life), therefore, the in vivo phenotype of slow clearance defines the reduced susceptibility to the drug. Measurement of parasite counts every six hours during the first three days after treatment have been recommended to measure the parasite clearance half-life, but it remains unclear whether simpler sampling intervals and frequencies might also be sufficient to reliably estimate this parameter. METHODS: A total of 2,746 parasite density-time profiles were selected from 13 clinical trials in Thailand, Cambodia, Mali, Vietnam, and Kenya. In these studies, parasite densities were measured every six hours until negative after treatment with an artemisinin derivative (alone or in combination with a partner drug). The WWARN Parasite Clearance Estimator (PCE) tool was used to estimate "reference" half-lives from these six-hourly measurements. The effect of four alternative sampling schedules on half-life estimation was investigated, and compared to the reference half-life (time zero, 6, 12, 24 (A1); zero, 6, 18, 24 (A2); zero, 12, 18, 24 (A3) or zero, 12, 24 (A4) hours and then every 12 hours). Statistical bootstrap methods were used to estimate the sampling distribution of half-lives for parasite populations with different geometric mean half-lives. A simulation study was performed to investigate a suite of 16 potential alternative schedules and half-life estimates generated by each of the schedules were compared to the "true" half-life. The candidate schedules in the simulation study included (among others) six-hourly sampling, schedule A1, schedule A4, and a convenience sampling schedule at six, seven, 24, 25, 48 and 49 hours. RESULTS: The median (range) parasite half-life for all clinical studies combined was 3.1 (0.7-12.9) hours. Schedule A1 consistently performed the best, and schedule A4 the worst, both for the individual patient estimates and for the populations generated with the bootstrapping algorithm. In both cases, the differences between the reference and alternative schedules decreased as half-life increased. In the simulation study, 24-hourly sampling performed the worst, and six-hourly sampling the best. The simulation study confirmed that more dense parasite sampling schedules are required to accurately estimate half-life for profiles with short half-life (≤ three hours) and/or low initial parasite density (≤ 10,000 per µL). Among schedules in the simulation study with six or fewer measurements in the first 48 hours, a schedule with measurements at times (time windows) of 0 (0-2), 6 (4-8), 12 (10-14), 24 (22-26), 36 (34-36) and 48 (46-50) hours, or at times 6, 7 (two samples in time window 5-8), 24, 25 (two samples during time 23-26), and 48, 49 (two samples during time 47-50) hours, until negative most accurately estimated the "true" half-life. For a given schedule, continuing sampling after two days had little effect on the estimation of half-life, provided that adequate sampling was performed in the first two days and the half-life was less than three hours. If the measured parasitaemia at two days exceeded 1,000 per µL, continued sampling for at least once a day was needed for accurate half-life estimates. CONCLUSIONS: This study has revealed important insights on sampling schedules for accurate and reliable estimation of Plasmodium falciparum half-life following treatment with an artemisinin derivative (alone or in combination with a partner drug). Accurate measurement of short half-lives (rapid clearance) requires more dense sampling schedules (with more than twice daily sampling). A more intensive sampling schedule is, therefore, recommended in locations where P. falciparum susceptibility to artemisinins is not known and the necessary resources are available. Counting parasite density at six hours is important, and less frequent sampling is satisfactory for estimating long parasite half-lives in areas where artemisinin resistance is present.

The quality of antimalarial medicines in western Cambodia: a case study along the Thai-Cambodian border.

The prevalence, availability, and use of antimalarial medicines (AMLs) were studied in six Cambodian provinces along the Thai-Cambodian border. The study was divided into two parts: the first looked at the quality of AMLs available in Pursat, Pailin, Battambang, Bantey Meanchey, Oddar Meanchey, and Preah Vihear and the second obtained information about the availability and use of AMLs. A randomized sampling methodology was used to select locations and collect samples, which were screened using Global Pharma Health Fund (GPHF) Minilabs. A subset of samples was sent to quality control laboratories for confirmatory testing. For the second part of the study, face-to-face interviews were conducted using standardized surveys with members of randomly selected households and staff of health facilities in the villages with highest malaria incidence to find out where they acquired their AMLs and which were most frequently used. The results showed an overall failure rate of 12.3% (n = 46 of 374 total AML samples). The causes of medication sample failure were low active pharmaceutical ingredient (API) content, failed dissolution properties, and unacceptably high levels of impurities. A total of 86.2% of survey respondents (n = 1,648 of 1,912) reported a member of their household having malaria in the previous year. The most commonly used medicines were paracetamol (67.1% of respondents), Malarine (A+M co-blistered, 28.6%), artesunate + mefloquine co-blistered (public sector product, 17.3%), quinine (16.7%), and artesunate monotherapy (11.9%). Health staff typically prescribed co-blistered artesunate plus mefloquine in the public sector (67.8%), the artesunate plus mefloquine "social marketing" product from Population Services International (PSI), Malarine (50.3%) in the private sector, artemether (49.7%), chloroquine (39%) and paracetamol (72.9%) to reduce fever.

Effect of high-dose or split-dose artesunate on parasite clearance in 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.

Genetic loci associated with delayed clearance of Plasmodium falciparum following artemisinin treatment in Southeast Asia.

The recent emergence of artemisinin-resistant Plasmodium falciparum malaria in western Cambodia could threaten prospects for malaria elimination. Identification of the genetic basis of resistance would provide tools for molecular surveillance, aiding efforts to contain resistance. Clinical trials of artesunate efficacy were conducted in Bangladesh, in northwestern Thailand near the Myanmar border, and at two sites in western Cambodia. Parasites collected from trial participants were genotyped at 8,079 single nucleotide polymorphisms (SNPs) using a P. falciparum-specific SNP array. Parasite genotypes were examined for signatures of recent positive selection and association with parasite clearance phenotypes to identify regions of the genome associated with artemisinin resistance. Four SNPs on chromosomes 10 (one), 13 (two), and 14 (one) were significantly associated with delayed parasite clearance. The two SNPs on chromosome 13 are in a region of the genome that appears to be under strong recent positive selection in Cambodia. The SNPs on chromosomes 10 and 13 lie in or near genes involved in postreplication repair, a DNA damage-tolerance pathway. Replication and validation studies are needed to refine the location of loci responsible for artemisinin resistance and to understand the mechanism behind it; however, two SNPs on chromosomes 10 and 13 may be useful markers of delayed parasite clearance in surveillance for artemisinin resistance in Southeast Asia.

Prevalence of Opisthorchis viverrini infection in humans and fish in Kratie Province, Cambodia.

Opisthorchis viverrini is a medically important foodborne parasite in the Indochina Peninsula. In Cambodia, the prevalence of this trematode has been reported in Takeo Province, but not in other areas. In this study, we investigated the prevalence of O. viverrini infection among people in seven riparian villages along the Mekong River, Kratie Province. We also examined the status of metacercarial infection in fish hosts. Fecal specimens were collected from 2101 residents and schoolchildren, and were examined by the Kato-Katz technique. The average O. viverrini egg positive rate was 4.6%, with the highest prevalence found in Roka Kandal A village (10.4%) followed by Talous village (5.9%). In these villages, adult residents showed higher prevalences (19.4% and 9.0%, respectively) than schoolchildren (6.4% and 1.4%, respectively). O. viverrini adult worms were recovered from 2 egg-positive cases (18 and 4 specimens) after praziquantel treatment and purgation. In addition, three of seven freshwater fish species caught near the villages were positive for O. viverrini metacercariae. A total of 367 metacercariae were harvested from 19 infected fish (metacercarial density; 19 per fish). The species of the metacercariae was confirmed through adult worm recovery by experimental infection to hamsters. The results provide evidence that the surveyed areas of Kratie Province, Cambodia, are endemic for O. viverrini infection.

Optimizing the HRP-2 in vitro malaria drug susceptibility assay using a reference clone to improve comparisons of Plasmodium falciparum field isolates.

BACKGROUND: Apparent emerging artemisinin-resistant Plasmodium falciparum malaria in Southeast Asia requires development of practical tools to monitor for resistant parasites. Although in vitro anti-malarial susceptibility tests are widely used, uncertainties remain regarding interpretation of P. falciparum field isolate values. METHODS: Performance parameters of the W2 P. falciparum clone (considered artemisinin "sensitive") were evaluated as a reference for the HRP-2 immediate ex vivo assay. Variability in W2 IC50s was assessed, including intra- and inter-assay variability among and between technicians in multiple experiments, over five freeze-thaw cycles, over five months of continuous culture, and before and after transport of drug-coated plates to remote field sites. Nominal drug plate concentrations of artesunate (AS) and dihydroartemisinin (DHA) were verified by LC-MS analysis. Plasmodium falciparum field isolate IC50s for DHA from subjects in an artemisinin-resistant area in Cambodia were compared with W2 susceptibility. RESULTS: Plate drug concentrations and day-to-day technical assay performance among technicians were important sources of variability for W2 IC50s within and between assays. Freeze-thaw cycles, long-term continuous culture, and transport to and from remote sites had less influence. Despite variability in W2 susceptibility, the median IC50s for DHA for Cambodian field isolates were higher (p <0.0001) than the W2 clone (3.9 nM), both for subjects with expected (less than 72 hours; 6.3 nM) and prolonged (greater or equal to 72 hours; 9.6 nM) parasite clearance times during treatment with artesunate monotherapy. CONCLUSION: The W2 reference clone improved the interpretability of field isolate susceptibility from the immediate ex vivo HRP-2 assay from areas of artemisinin resistance. Methods to increase the reproducibility of plate coating may improve overall assay interpretability and utility.

Optimising strategies for Plasmodium falciparum malaria elimination in Cambodia: primaquine, mass drug administration and artemisinin resistance.

BACKGROUND: Malaria elimination requires a variety of approaches individually optimized for different transmission settings. A recent field study in an area of low seasonal transmission in South West Cambodia demonstrated dramatic reductions in malaria parasite prevalence following both mass drug administration (MDA) and high treatment coverage of symptomatic patients with artemisinin-piperaquine plus primaquine. This study employed multiple combined strategies and it was unclear what contribution each made to the reductions in malaria. METHOD AND FINDINGS: A mathematical model fitted to the trial results was used to assess the effects of the various components of these interventions, design optimal elimination strategies, and explore their interactions with artemisinin resistance, which has recently been discovered in Western Cambodia. The modelling indicated that most of the initial reduction of P. falciparum malaria resulted from MDA with artemisinin-piperaquine. The subsequent continued decline and near elimination resulted mainly from high coverage with artemisinin-piperaquine treatment. Both these strategies were more effective with the addition of primaquine. MDA with artemisinin combination therapy (ACT) increased the proportion of artemisinin resistant infections, although much less than treatment of symptomatic cases with ACT, and this increase was slowed by adding primaquine. Artemisinin resistance reduced the effectiveness of interventions using ACT when the prevalence of resistance was very high. The main results were robust to assumptions about primaquine action, and immunity. CONCLUSIONS: The key messages of these modelling results for policy makers were: high coverage with ACT treatment can produce a long-term reduction in malaria whereas the impact of MDA is generally only short-term; primaquine enhances the effect of ACT in eliminating malaria and reduces the increase in proportion of artemisinin resistant infections; parasite prevalence is a better surveillance measure for elimination programmes than numbers of symptomatic cases; combinations of interventions are most effective and sustained efforts are crucial for successful elimination.

High prevalence of Opisthorchis viverrini infection in a riparian population in Takeo Province, Cambodia.

Opisthorchis viverrini infection was found to be highly prevalent in 3 riverside villages (Ang Svay Chek A, B, and C) of the Prey Kabas District, Takeo Province. This area is located in the southern part of Cambodia, where the recovery of adult O. viverrini worms was recently reported. From May 2006 until May 2010, fecal examinations were performed on a total of 1,799 villagers using the Kato-Katz thick smear technique. In the 3 villages, the overall positive rate for helminth eggs ranged from 51.7 to 59.0% (av. 57.4%), and the percentage positive for O. viverrini was 46.4-50.6% (47.5%). Other helminths detected included hookworms (13.2%), echinostomes (2.9%), Trichuris trichiura (1.3%), Ascaris lumbricoides (0.6%), and Taenia spp. (0.06%). The prevalence of O. viverrini eggs appeared to reflect a lower infection in younger individuals (<20 years) than in the adult population (>20 years). Men (50.4%) revealed a significantly higher (P=0.02) prevalence than women (44.3%). The Ang Svay Chek villages of the Prey Kabas District, Takeo Province, Cambodia have been confirmed to be a highly endemic area for human O. viverrini infection.

Analysis of Plasmodium falciparum diversity in natural infections by deep sequencing.

Malaria elimination strategies require surveillance of the parasite population for genetic changes that demand a public health response, such as new forms of drug resistance. Here we describe methods for the large-scale analysis of genetic variation in Plasmodium falciparum by deep sequencing of parasite DNA obtained from the blood of patients with malaria, either directly or after short-term culture. Analysis of 86,158 exonic single nucleotide polymorphisms that passed genotyping quality control in 227 samples from Africa, Asia and Oceania provides genome-wide estimates of allele frequency distribution, population structure and linkage disequilibrium. By comparing the genetic diversity of individual infections with that of the local parasite population, we derive a metric of within-host diversity that is related to the level of inbreeding in the population. An open-access web application has been established for the exploration of regional differences in allele frequency and of highly differentiated loci in the P. falciparum genome.

Using CF11 cellulose columns to inexpensively and effectively remove human DNA from Plasmodium falciparum-infected whole blood samples.

BACKGROUND: Genome and transcriptome studies of Plasmodium nucleic acids obtained from parasitized whole blood are greatly improved by depletion of human DNA or enrichment of parasite DNA prior to next-generation sequencing and microarray hybridization. The most effective method currently used is a two-step procedure to deplete leukocytes: centrifugation using density gradient media followed by filtration through expensive, commercially available columns. This method is not easily implemented in field studies that collect hundreds of samples and simultaneously process samples for multiple laboratory analyses. Inexpensive syringes, hand-packed with CF11 cellulose powder, were recently shown to improve ex vivo cultivation of Plasmodium vivax obtained from parasitized whole blood. This study was undertaken to determine whether CF11 columns could be adapted to isolate Plasmodium falciparum DNA from parasitized whole blood and achieve current quantity and purity requirements for Illumina sequencing. METHODS: The CF11 procedure was compared with the current two-step standard of leukocyte depletion using parasitized red blood cells cultured in vitro and parasitized blood obtained ex vivo from Cambodian patients with malaria. Procedural variations in centrifugation and column size were tested, along with a range of blood volumes and parasite densities. RESULTS: CF11 filtration reliably produces 500 nanograms of DNA with less than 50% human DNA contamination, which is comparable to that obtained by the two-step method and falls within the current quality control requirements for Illumina sequencing. In addition, a centrifuge-free version of the CF11 filtration method to isolate P. falciparum DNA at remote and minimally equipped field sites in malaria-endemic areas was validated. CONCLUSIONS: CF11 filtration is a cost-effective, scalable, one-step approach to remove human DNA from P. falciparum-infected whole blood samples.

Scale-up of community-based malaria control can be achieved without degrading community health workers' service quality: the Village Malaria Worker project in Cambodia.

BACKGROUND: Malaria control has been scaled up in many developing countries in their efforts to achieve the Millennium Development Goals. Cambodia recently scaled up their Village Malaria Worker (VMW) project by substantially increasing the number of VMWs and expanding the project's health services to include treatment of fever, diarrhoea, and Acute Respiratory Infections (ARI) in children under five. This study examined if the scale-up interfered with VMWs' service quality, actions, and knowledge of malaria control, and analysed VMWs' overall achievements and perceptions of the newly added health services. METHODS: Structured interviews were conducted pre scale-up in February-March 2008 with 251 VMWs and post scale-up in July-August 2010 with 252 VMWs. Comparing the pre and post scale-up survey results (n = 195), changes were examined in terms of VMWs' 1) service quality, 2) malaria prevention and vector control actions, and 3) knowledge of malaria epidemiology and vector ecology. In addition, VMWs' newly added health services were descriptively analysed based on the post scale-up survey (n = 252). RESULTS: VMWs' service quality and actions significantly improved overall during the scale-up of the VMW project (mean index score: +0.805, p < 0.001; +2.923, p < 0.001; respectively). Although most of knowledge areas also showed significant improvement (between +0.256 and +0.499, p < 0.001), less than half (10.3%-47.7%) of the VMWs correctly answered a set of questions on malaria epidemiology and vector ecology, even in the post scale-up survey. About 70% of the respondents reported that their health services to control malaria remained the same or that they were more active after the scale-up. Two-thirds (66.3%) had become more enthusiastic about serving as a VMW since the scale-up, and all but one respondent reported being willing to continue the new services. CONCLUSIONS: The Cambodian experience clearly demonstrated that a nationwide scale-up of community-based malaria control can be achieved without degrading community health workers' service quality. The government's strategy to expand VMWs' health services, while providing sufficient training to maintain the quality of their original malaria control services, could have contributed to the improvement of VMW's service quality, actions, and knowledge in spite of the rapid scale-up of the project.

Case management of malaria fever in Cambodia: results from national anti-malarial outlet and household surveys.

BACKGROUND: Continued progress towards global reduction in morbidity and mortality due to malaria requires scale-up of effective case management with artemisinin-combination therapy (ACT). The first case of artemisinin resistance in Plasmodium falciparum was documented in western Cambodia. Spread of artemisinin resistance would threaten recent gains in global malaria control. As such, the anti-malarial market and malaria case management practices in Cambodia have global significance. METHODS: Nationally-representative household and outlet surveys were conducted in 2009 among areas in Cambodia with malaria risk. An anti-malarial audit was conducted among all public and private outlets with the potential to sell anti-malarials. Indicators on availability, price and relative volumes sold/distributed were calculated across types of anti-malarials and outlets. The household survey collected information about management of recent "malaria fevers." Case management in the public versus private sector, and anti-malarial treatment based on malaria diagnostic testing were examined. RESULTS: Most public outlets (85%) and nearly half of private pharmacies, clinics and drug stores stock ACT. Oral artemisinin monotherapy was found in pharmacies/clinics (9%), drug stores (14%), mobile providers (4%) and grocery stores (2%). Among total anti-malarial volumes sold/distributed nationally, 6% are artemisinin monotherapies and 72% are ACT. Only 45% of people with recent "malaria fever" reportedly receive a diagnostic test, and the most common treatment acquired is a drug cocktail containing no identifiable anti-malarial. A self-reported positive diagnostic test, particularly when received in the public sector, improves likelihood of receiving anti-malarial treatment. Nonetheless, anti-malarial treatment of reportedly positive cases is low among people who seek treatment exclusively in the public (61%) and private (42%) sectors. CONCLUSIONS: While data on the anti-malarial market shows favourable progress towards replacing artemisinin monotherapies with ACT, the widespread use of drug cocktails to treat malaria is a barrier to effective case management. Significant achievements have been made in availability of diagnostic testing and effective treatment in the public and private sectors. However, interventions to improve case management are urgently required, particularly in the private sector. Evidence-based interventions that target provider and consumer behaviour are needed to support uptake of diagnostic testing and treatment with full-course first-line anti-malarials.

Comparative phylogeography reveals a shared impact of pleistocene environmental change in shaping genetic diversity within nine Anopheles mosquito species across the Indo-Burma biodiversity hotspot.

South-East Asia is one of the world's richest regions in terms of biodiversity. An understanding of the distribution of diversity and the factors shaping it is lacking, yet essential for identifying conservation priorities for the region's highly threatened biodiversity. Here, we take a large-scale comparative approach, combining data from nine forest-associated Anopheles mosquito species and using statistical phylogeographical methods to disentangle the effects of environmental history, species-specific ecology and random coalescent effects. Spatially explicit modelling of Pleistocene demographic history supports a common influence of environmental events in shaping the genetic diversity of all species examined, despite differences in species' mtDNA gene trees. Populations were periodically restricted to allopatric northeastern and northwestern refugia, most likely due to Pleistocene forest fragmentation. Subsequent southwards post-glacial recolonization is supported by a north-south gradient of decreasing genetic diversity. Repeated allopatric fragmentation and recolonization have led to the formation of deeply divergent geographical lineages within four species and a suture zone where these intraspecific lineages meet along the Thai-Myanmar border. A common environmental influence for this divergence was further indicated by strong support for simultaneous divergence within the same four species, dating to approximately 900 thousand years ago (kya). Differences in the geographical structuring of genetic diversity between species are probably the result of varying species' biology. The findings have important implications for conservation planning; if the refugial regions and suture zone identified here are shared by other forest taxa, the unique and high levels of genetic diversity they house will make these areas conservation priorities.