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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.

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 (IC 50 ) 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 IC 50 s 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 (IC 50 s 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.

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.

Artemisinin-resistant Plasmodium falciparum malaria has emerged in western Cambodia. Resistance is characterized by prolonged in vivo parasite clearance times (PCTs) following artesunate treatment. The biological basis is unclear. The hypothesis that delayed parasite clearance results from a stage-specific reduction in artemisinin sensitivity of the circulating young asexual parasite ring stages was examined. A mathematical model was developed, describing the intrahost parasite stage-specific pharmacokinetic–pharmacodynamic relationships. Model parameters were estimated using detailed pharmacokinetic and parasite clearance data from 39 patients with uncomplicated falciparum malaria treated with artesunate from Pailin (western Cambodia) where artemisinin resistance was evident and 40 patients from Wang Pha (northwestern Thailand) where efficacy was preserved. The mathematical model reproduced the observed parasite clearance for each patient with an accurate goodness of fit (rmsd: 0.03–0.67 in log 10 scale). The parameter sets that provided the best fits with the observed in vivo data consist of a highly conserved concentration–effect relationship for the trophozoite and schizont parasite stages, but a variable relationship for the ring stages. The model-derived assessment suggests that the efficacy of artesunate on ring stage parasites is reduced significantly in Pailin. This result supports the hypothesis that artemisinin resistance mainly reflects reduced ring-stage susceptibility and predicts that doubling the frequency of dosing will accelerate clearance of artemisinin-resistant parasites.

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.

In western Cambodia, malaria parasites clear slowly from the blood after treatment with artemisinin derivatives, but it is unclear whether this results from parasite, host, or other factors specific to this population. We measured heritability of clearance rate by evaluating patients infected with identical or nonidentical parasite genotypes, using methods analogous to human twin studies. A substantial proportion (56%-58%) of the variation in clearance rate is explained by parasite genetics. This has 2 important implications: (1) selection with artemisinin derivatives will tend to drive resistance spread and (2) because heritability is high, the genes underlying parasite clearance rate may be identified by genome-wide association.

Background Artemisinin resistance in Plasmodium falciparum malaria has emerged in Western Cambodia. This is a major threat to global plans to control and eliminate malaria as the artemisinins are a key component of antimalarial treatment throughout the world. To identify key features associated with the delayed parasite clearance phenotype, we employed DNA microarrays to profile the physiological gene expression pattern of the resistant isolates. Results In the ring and trophozoite stages, we observed reduced expression of many basic metabolic and cellular pathways which suggests a slower growth and maturation of these parasites during the first half of the asexual intraerythrocytic developmental cycle (IDC). In the schizont stage, there is an increased expression of essentially all functionalities associated with protein metabolism which indicates the prolonged and thus increased capacity of protein synthesis during the second half of the resistant parasite IDC. This modulation of the P. falciparum intraerythrocytic transcriptome may result from differential expression of regulatory proteins such as transcription factors or chromatin remodeling associated proteins. In addition, there is a unique and uniform copy number variation pattern in the Cambodian parasites which may represent an underlying genetic background that contributes to the resistance phenotype. Conclusions The decreased metabolic activities in the ring stages are consistent with previous suggestions of higher resilience of the early developmental stages to artemisinin. Moreover, the increased capacity of protein synthesis and protein turnover in the schizont stage may contribute to artemisinin resistance by counteracting the protein damage caused by the oxidative stress and/or protein alkylation effect of this drug. This study reports the first global transcriptional survey of artemisinin resistant parasites and provides insight to the complexities of the molecular basis of pathogens with drug resistance phenotypes in vivo.

International audience; 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.

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). 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.

Following preventive chemotherapy covering the entire population in the two endemic regions in Cambodia, the prevalence of schistosomiasis dropped from 77% in 1995 to 0.5% in 2003. The study presented here reports on the running cost of the control programme, and evaluates its cost-effectiveness and cost-benefit. Financial costs were assessed using data taken from the annual reports of the National Center for Malaria Control, the Cambodian institution responsible for the control activities. Other data were collected from interviews with provincial and district staff. The analysis was conducted from the point of views of the Cambodian Ministry of Health and that of the society, and the comparison was undertaken using the "do-nothing" option. The cost to treat an individual for the 9 years period of the implementation phase was 9.22 USD (1.02 per year), the cost for each severe infection avoided was 61.50 USD and 6531 USD for each death avoided. The drug cost corresponds on average to 17.34% of the programme's implementation cost. The cost of bringing one severely infected individual of productive age to complete productivity, was estimated at 114 USD and for 1 USD invested in the programme the return in increased productivity, for the economic system, was estimated to be 3.85 USD. The control programme demonstrated significant economical advantages. However, its costs are too high to be entirely supported by the Cambodian Ministry of Health.

Antimalarial drugs impose strong selective pressure on Plasmodium falciparum parasites and leave signatures of selection in the parasite genome; screening for genes under selection may suggest potential drug or immune targets. Genome-wide association studies (GWAS) of parasite traits have been hampered by the lack of high-throughput genotyping methods, inadequate knowledge of parasite population history and time-consuming adaptations of parasites to in vitro culture. Here we report the first Plasmodium GWAS, which included 189 culture-adapted P. falciparum parasites genotyped using a custom-built Affymetrix molecular inversion probe 3K malaria panel array with a coverage of approximately 1 SNP per 7 kb. Population structure, variation in recombination rate and loci under recent positive selection were detected. Parasite half-maximum inhibitory concentrations for seven antimalarial drugs were obtained and used in GWAS to identify genes associated with drug responses. This study provides valuable tools and insight into the P. falciparum genome.

Aedes aegypti mosquitoes originated in Africa and are thought to have spread recently to Southeast Asia, where they are the major vector of dengue. Thirteen microsatellite loci were used to determine the genetic population structure of A. aegypti at a hierarchy of spatial scales encompassing 36 sites in Myanmar, Cambodia and Thailand, and two sites in Sri Lanka and Nigeria. Low, but significant, genetic structuring was found at all spatial scales (from 5 to >2000 km) and significant FIS values indicated genetic structuring even within 500 m. Spatially dependent genetic-clustering methods revealed that although spatial distance plays a role in shaping larger-scale population structure, it is not the only factor. Genetic heterogeneity in major port cities and genetic similarity of distant locations connected by major roads, suggest that human transportation routes have resulted in passive long-distance migration of A. aegypti. The restricted dispersal on a small spatial scale will make localized control efforts and sterile insect technology effective for dengue control. Conversely, preventing the establishment of insecticide resistance genes or spreading refractory genes in a genetic modification strategy would be challenging. These effects on vector control will depend on the relative strength of the opposing effects of passive dispersal.

Summary Objectives To identify and describe the distribution of dengue vectors and factors affecting this distribution in Cambodia, with a view to practicing rational, evidence-based dengue outbreak prevention activities. Methods Entomological survey with a questionnaire component in 100 randomly selected households in each of 13 clusters of high or low human population density of seven Cambodian provinces. Entomological and other indices were calculated, and statistical methods used to describe factors of potential outbreak risk. Results Aedes aegypti was the principle dengue vector in all clusters, making up 95.5% (20 555 of 21 325) of the Aedes pupae population. The majority of pupae were recovered either from large concrete water storage jars (16 230; 76.1%) or concrete water storage tanks (2819; 13.2%). There were small but significantly higher levels of dengue vector infestation in rural than urban areas. The mean pupae density over the survey was 16.4/house, which ranged between clusters from 5.2/house to 56.9/house. The ‘pupae-per-person’ index was 2.4 and 3.6 in urban and rural areas, respectively, and was independent of mean human population density or household water container distribution. Conclusions High populations of household-associated dengue vectors were present in all surveyed clusters. The highly skewed distribution of pupae in a limited number of key containers suggests adoption and further development of community-based control measures targeting these containers holds most potential chance of controlling dengue outbreaks in Cambodia.

Summary Objective To assess the cost-effectiveness (CE) of annual targeted larviciding campaigns from 2001 to 2005 against the dengue vector Aedes aegypti in two urban areas of Cambodia with a population of 2.9 million people. Methods The intervention under analysis consisted of annual larviciding campaigns targeting medium to large water storage containers in households and other premises. The CE compared the intervention against the hypothetical alternative of no intervention. The CE was calculated as the ratio of disability adjusted life years (DALYs) saved to the net cost of the intervention (in 2005 US dollars) by year. A sensitivity analysis explored the range of study parameters. Results The intervention reduced the number of dengue cases and deaths by 53%. It averted an annual average of 2980 dengue hospitalizations, 11 921 dengue ambulatory cases and 23 dengue deaths, resulting in a saving of 997 DALYs per year. The gross cost of the intervention was US$ 567 800 per year, or US$ 0.20 per person covered. As the intervention averted considerable medical care, the annual net cost of the intervention was US$ 312 214 (US$ 0.11 per person covered) from a public sector perspective and US$ 37 137 (US$ 0.01 per person covered) from a societal perspective. The resulting CE ratios were: US$ 313/DALY gained from the public perspective and US$ 37/DALY gained from the societal perspective. Even under the most conservative assumption, the intervention remained cost effective from both perspectives. Conclusions Annual, targeted larviciding campaigns appear to have been effective and cost-effective medium-term interventions to reduce the epidemiologic and economic burden of dengue in urban areas of Cambodia. Objectif Evaluer la rentabilite des campagnes annuelles utilisant des larvicides contre Aedes aegypti le vecteur de la dengue de 2001 a 2005 dans deux regions urbaines du Cambodge avec une population de 2,9 millions d’habitants. Methodes L’intervention analysee consistait en des campagnes annuelles utilisant des larvicides sur des moyens et grands recipients d’eau dans les menages et autres premisses. La mesure de la rentabilite a compare l’intervention par rapport a l’alternative hypothetique de non intervention. La rentabilite a ete calculee comme etant le rapport entre les annees de vie sauvees ajustees par l’incapacite (DALY) et le cout net de l’intervention par annee (valeur du dollars USA de 2005). Une analyse de sensibilite a explore la gamme des parametres d’etude. Resultats L’intervention a reduit le nombre de cas et de deces de la dengue de 53%. Elle a evite en moyenne 2980 hospitalisations, 11 921 cas ambulatoires et 23 deces par la dengue, avec pour resultat une economie de 997 DALY par an. Le cout brut de l’intervention etait de 567 800 dollars US par an soit 0,20 dollars US par personne couvert. Puisque l’intervention a evite un nombre considerable de soins medicaux, le cout net de l’intervention etait de 312 214 dollars US (0,11 dollars US par personne couvert) pour une perspective de secteur publique et de 37 137 dollars US (0,01 dollars US par personne couvert) pour une perspective sociale. Les rapports rentabilite resultantes etaient: 313 dollars US/DALY selon la perspective publique et de 37 dollars US/DALY selon la perspective sociale. Meme avec l’assomption la plus conservatrice, l’intervention est demeuree rentable selon les deux perspectives. Conclusions Les campagnes annuelles utilisant des larvicides semblent avoir ete des interventions efficaces et rentables a moyen terme pour reduire la charge epidemiologique et economique de la dengue dans des regions urbaines du Cambodge. Objetivo Evaluar la costo-efectividad de las campanas anuales de aplicacion de larvicidas contra el vector del dengue Aedes aegypti desde el 2001 y hasta el 2005 en dos areas urbanas de Cambodia con una poblacion de 2.9 millones de personas. Metodos La intervencion analizada consistia en campanas anuales de aplicacion de larvicidas sobre contenedores medianos a grandes utilizados para el almacenamiento de agua en hogares y otros lugares. La costo-efectividad (CE) comparo la intervencion frente a la alternativa hipotetica de no intervencion. La CE se calculo como los anos de vida ajustados por discapacidad (DALYs) ganados al costo neto de la intervencion (en dolares americanos del 2005) por ano. Un analisis de sensibilidad exploro el rango de los parametros del estudio. Resultados La intervencion redujo el numero de casos de dengue y de muertes en un 53%. Previno un promedio de 2980 hospitalizaciones por dengue, 11 921 casos ambulatorios de dengue, y 23 muertes por dengue, resultando en una ganancia de DALYs por ano. El coste bruto de la intervencion fue de US$ 567 800 por ano, o US$ 0.20 por persona cubierta. Puesto que la intervencion previno un cuidado medico considerable, el coste neto de la intervencion fue de US$ 312 214 (US$ 0.11 por persona cubierta) desde una perspectiva del sector publico y US$ 37 137 (US$ 0.01 por persona cubierta) desde una perspectiva social. Las proporciones de coste-efectividad resultantes fueron: US$ 313/DALY ganados desde una perspectiva publica y US$ 37/DALY ganados desde una perspectiva social. Aun bajo las asunciones mas conservadoras, la intervencion era costo efectiva desde ambas perspectivas. Conclusiones Las campanas anuales y dirigidas con larvicidas parecen haber sido efectivas, siendo intervenciones costo efectivas a medio plazo para reducir la carga epidemiologica y economica del dengue en areas urbanas de Cambodia.

Summary Objectives To compare the efficacy and tolerability of dihydroartemisinin–piperaquine (DHA–PQP) with that of a 3-day regimen of mefloquine and artesunate (MAS3) for the treatment of uncomplicated falciparum malaria in Cambodia. Method Randomized open-label non-inferiority study over 64 days. Results Four hundred and sixty-four patients were included in the study. The polymerase chain reaction genotyping-adjusted cure rates on day 63 were 97.5% (95% confidence interval, CI, 93.8–99.3) for DHA–PQP and 97.5% (95% CI, 93.8–99.3) for MAS3, P = 1. There were no serious adverse events, but significantly more episodes of vomiting (P = 0.03), dizziness (P = 0.002), palpitations (P = 0.04), and sleep disorders (P = 0.03) reported in the MAS3 treatment group, consistent with the side-effect profile of mefloquine. Conclusions DHA–PQP was as efficacious as MAS3, but much better tolerated, making it more appropriate for use in a routine programme setting. This highly efficacious, safe and more affordable fixed-dose combination could become the treatment of choice for Plasmodium falciparum malaria in Cambodia. Objectifs Comparer l'efficacite et la tolerance du dihydroartemisinine-piperaquine (DHA–PQP) a celles d'un regime a base de mefloquine de trois jours (MAS3) pour le traitement de la malaria falciparum non compliquee au Cambodge. Methode Etude Randomisee ouverte de non inferiorite sur 64 jours. Resultats 464 patients ont ete inclus dans l’etude. Les taux guerison au jour 63 ajustes par les resultats du genotypage par la reaction en chaine de la polymerase etaient de 97,5% (IC95%: 93.8–99.3) pour le DHA–PQP et de 97,5% (IC95%: 93,8–99,3) pour le MAS3, P = 1. Il n'y avait aucun effet adverse serieux, mais de facon significative, des episodes de vomissement (P = 0,03), des vertiges (P = 0,002), des palpitations (P = 0,04), et des troubles de sommeil (P = 0.03) ont ete rapportes dans le groupe du traitement au MAS3, ce qui etait consistant avec les profiles d'effets secondaires du mefloquine. Conclusion le DHA–PQP etait aussi efficace que le MAS3, mais bien mieux tolere, le rendant ainsi plus approprie pour l'usage en routine dans le cadre d'un programme. Cette combinaison a dose fixe de grande efficacite, sure et plus accessible pourrait devenir le traitement de choix pour la malaria aPlasmodium falciparum au Cambodge. Objetivos Comparar la eficacia y la tolerabilidad de la dihidroartemisinina-piperaquina (DHA–PQP) con la de un regimen de 3 dias de mefloquina (MAS3), para el tratamiento de la malaria no complicada por falciparum en Cambodia Metodo Estudio aleatorizado, abierto, de no-inferioridad, durante 64 dias. Resultados Se incluyeron 464 pacientes en el estudio. Las tasas de curacion en el dia 63, ajustadas por genotipaje mediante PCR, fueron del 97.5% (95% IC: 93.8–99.3) para DHA–PQP y del 97.5% (95%IC: 93.8–99.3) para MAS3, P = 1. No se observaron eventos adversos serios, pero si se reporto un numero significativo de episodios de vomitos (P = 0.03), mareos (P = 0.002), palpitaciones (P = 0.04), y desordenes del sue,no (P = 0.03) entre el grupo de tratamiento con MAS3, algo consistente con el perfil de efectos secundarios de la mefloquina. Conclusiones La DHA–PQP fue tan eficaz como la MAS3, ademas de ser mejor tolerada, siendo mas apropiada para el uso dentro del marco de un programa de rutina. Esta combinacion de dosis fija, altamente eficaz, segura y mas asequible, podria convertirse en el tratamiento de eleccion para malaria por Plasmodium falciparum en Cambodia.

Summary In Cambodia, schistosomiasis is transmitted in the provinces of Kratie and Stung Treng where approximately 80 000 individuals are estimated to be at risk of infection. The baseline prevalence of infection was estimated to be between 73% and 88%, and cases of severe morbidity (hepatosplenomegaly, puberty retardation) and mortality were very common. In 1994, the Ministry of Health of Cambodia started schistosomiasis control applying universal chemotherapy with praziquantel (40 mg/kg). The coverage of the programme was between 62% and 86% for 8 years. This simple control measure resulted in the control of the disease: no cases were reported in 2004 and only three cases were reported in 2005. In addition, there are no longer reports of cases of severe morbidity due to schistosomiasis. Since the beginning of the control programme, a single dose of mebendazole (500 mg) has been combined with praziquantel during the mass chemotherapy; as a result the prevalence of Ascaris lumbricoides and hookworms dropped from 74.5% to 10% and from 86% to 40% respectively. The experience in Cambodia demonstrates that, with political commitment, control of parasitic diseases is achievable even in a situation of minimal resources. The programme represents a successful model for other developing countries.

Summary Objective To determine the efficacy of artemether–lumefantrine malaria treatment, as an alternative to artesunate + mefloquine, which is becoming ineffective in some areas of the Thai–Cambodian border. Methods Two studies were conducted to monitor the efficacy of artemether–lumefantrine in Sampov Lun referral hospital, Battambang Province, in 2002 and 2003, and one study was conducted to assess the efficacy of mefloquine + artesunate in 2003 for comparison. The studies were performed according to the WHO standardized protocol with a follow-up of 28 days. The therapeutic efficacy tests were complemented with in vitro tests and in 2003, with the measurement of lumefantrine plasma concentration at day 7 for the patients treated with artemether–lumefantrine. Results A total of 190 patients were included: 55 were treated with artemether–lumefantrine in 2002 (AL2002), 80 with artemether–lumefantrine and food supplementation in 2003 (AL2003) and 55 with artesunate + mefloquine in 2003 (AM2003). With the per-protocol analysis, the cure rate was 71.1% in study AL2002, 86.5% in study AL2003 and 92.4% in study AM2003. All the data were PCR corrected. The artemether–lumefantrine cure rate was unexpectedly low in 2002, but it increased with food supplementation in 2003. There was a significant difference (P = 0.02) in lumefantrine plasma concentrations between adequate clinical and parasitological responses and treatment failure cases. In vitro susceptibility to lumefantrine was reduced for isolates sampled from patients presenting with treatment failure, but the difference was not statistically different from isolates sampled from patients who were successfully treated. Conclusion Treatment failure cases of artemether–lumefantrine are most probably because of low levels of lumefantrine blood concentration. Further investigations are necessary to determine whether resistance of Plasmodium falciparum isolates to lumefantrine is present in the region. Objectif Determiner l'efficacite de l'artemether–lumefantrine pour le traitement paludisme en tant qu'alternative a l'artesunate + mefloquine qui devient de plus en plus inefficace dans certaines zones dans la frontiere Thailando–Cambodgienne. Methodes Deux etudes ont ete menees pour mesurer l'efficacite de l'artemether–lumefantrine dans l'hopital de reference de Sampov Lun dans la province de Battambang en 2002 et 2003 et, dans un but de comparaison, une etude a aussi ete menee pour evaluer l'efficacite de l'artesunate + mefloquine en 2003. Les etudes ont ete menees selon les protocoles standardises de l'OMS avec un suivi de 28 jours. Les tests d'efficacite therapeutique ont ete complementes par des tests in vitro et, en 2003 par la mesure de la concentration plasmatique de lumefantrine au jour 7 pour les patients traites a l'artemether–lumefantrine. Resultats Au total 190 patients ont ete inclus dans l’etude parmi lesquels 55 ont ete traites a l'artemether–lumefantrine en 2002 (AL2002), 80 a l'artemether–lumefantrine en plus d'un complement de nourriture en 2003 (AL2003) et 55 a l'artesunate + mefloquine en 2003 (AM2003). L'analyse suivant un protocole standard a revele un taux de guerison de 71,1% pour AL2002, 86,5% pour AL2003 et 92,4% pour AM2003. Toutes les donnees ont ete ajustees avec les resultats de la PCR. Le taux de guerison pour l'artemether–lumefantrine s'est avere bas de facon inattendue en 2002 mais il a augmente avec le complement de nourriture en 2003. Il y avait une difference significative (p = 0,02) pour les concentrations plasmatiques de lumefantrine entre d'une part les reponses cliniques et parasitologiques adequates et d'autre part les cas d’echec therapeutique. La susceptibilite in vitro pour la lumefantrine etait reduite pour les souches isolees de patients presentant un echec therapeutique mais les valeurs n’etaient pas statistiquement differentes de celles de souches isolees de patients traites avec succes. Conclusion Les cas d’echec therapeutique a l'artemether–lumefantrine sont plus probablement dus a des concentrations sanguines basses de lumefantrine. Des investigations supplementaires sont necessaires pour determiner s'il existe une resistance de P. falciparuma la lumefantrine dans la region. Objetivo Determinar la eficacia del tratamiento de malaria con artemeter-lumefantrina como alternativa al artesunato + mefloquina, el cual esta dejando de ser efectivo en algunas areas de la frontera entre Tailanda y Cambodia. Metodos Se llevaron a cabo dos estudios para monitorizar la eficacia de artemeter-lumefantrina en el hospital de referencia de Sampov Lun, provincia de Battambang, entre el 2002 y 2003, asi como un estudio durante el 2003, y a modo de comparacion, para evaluar la eficacia de la mefloquina + artesunato. Estos estudios se realizaron siguiendo el protocolo estandar de la OMS con un seguimiento de 28 dias. Las pruebas de eficacia terapeutica se complementaron con pruebas in vitro y, durante el 2003, con la medicion de la concentracion de lumefantrina en plasma en el dia 7 para los pacientes tratados con artemeter-lumefantrina. Resultados Se incluyeron 190 pacientes: 55 fueron tratados con artemeter-lumefantrina en el 2002 (AL2002), 80 con artemeter-lumefantrina y suplementacion alimenticia en el 2003 (AL2003), y 55 con artesunato + mefloquina en el 2003. Con el analisis por protocolo, la tasa de curacion fue de 71.1% en el estudio AL2002, 86.5% en el estudio AL2003, y 92.4% en el estudio AM2003. Todos los datos fueron corregidos mediante PCR. La tasa de curacion para el tratamiento con artemeter-lumefantrina fue sorprendentemente baja en el 2002, pero aumento con la suplementacion alimenticia en el 2003. Se encontro una diferencia significativa (p = 0.02) en las concentraciones de lumefantrina en plasma entre casos con una respuesta clinica y parasitologica adecuadas y aquellos con fallo terapeutico. La susceptibilidad in vitro frente a la lumefantrina se redujo para cepas aisladas de pacientes que presentaban fallo en el tratamiento, pero la diferencia no era estadisticamente significativa de aquella para cepas aisladas de pacientes que hubiesen respondido adecuadamente al tratamiento. Conclusions Los casos de fallo terapeutico con artemeter-lumefantrina probablemente son debidos a bajos niveles de concentracion de lumefantrina en la sangre. Se necesitan mas estudios para determinar si existe resistencia a la lumefantrina entre las cepas de P. falciparum de la region.

We assessed the filariasis disease burden in four northeastern provinces of Cambodia by using and validating a key-informant questionnaire, consisting of four questions, with pictures of patients with leg elephantiasis and hydrocoele. The questionnaire was distributed and collected through the school, health and administrative systems. Validation surveys included clinical examination, a card test for W. bancrofti (ICT Filariasis card test, AMRAD) and night blood finger prick examination of patients reported with clinical elephantiasis. Only 48.0% of questionnaires were returned. A total of 220 patients were reported, mostly from Stung Treng (36.8%) and Rattanakiri provinces (35.0%). Key-informants reported patients with lymphatic filariasis with a sensitivity of 85.7% for leg and 97.0% for scrotum morbidity, and with a specificity of 95.6%. However, substantial over-reporting resulted in very low positive predictive values for elephantiasis of 19.4% for legs and of 23.7% for the scrotum. As 97.4% of patients with clinical lymphatic filariasis were older than 40 years, the diagnostic performance of the questionnaire would be improved by restricting its use to that age group. About 0.7% of 3490 W. bancrofti card tests were positive; the prevalence was 1.94% (12/618) in Rattanakiri, 0.38% (4/1055) in Stung Treng and 0.22% (2/919) in Preah Vihear. W. bancrofti microfilaria were identified in blood from two patients in Rattanakiri (0.32%) and from one patient in Stung Treng (0.09%). Brugia malayi microfilaria were identified in blood from five patients in Rattanakiri (0.81%) only. No patients with microfilariaemia were identified in Preah Vehear. In Mondulkiri province all investigations (card test, night blood examination, clinical examination) for lymphatic filariasis were negative. Our findings confirm the usefulness of key-informant questionnaire for the identification of filariasis patients provided that high adherence can be achieved. Lymphatic filariasis infection and disease is present in northern Cambodian provinces but the burdens of disease and infection are relatively low. These results are being used in the implementation of the national control programme for lymphatic filariasis.

Background Artemisinin resistance in Plasmodium falciparum manifests as slow parasite clearance but this measure is also influenced by host immunity, initial parasite biomass and partner drug efficacy. This study collated data from clinical trials of artemisinin derivatives in falciparum malaria with frequent parasite counts to provide reference parasite clearance estimates stratified by location, treatment and time, to examine host factors affecting parasite clearance, and to assess the relationships between parasite clearance and risk of recrudescence during follow-up. Methods Data from 24 studies, conducted from 1996 to 2013, with frequent parasite counts were pooled. Parasite clearance half-life (PC1/2) was estimated using the WWARN Parasite Clearance Estimator. Random effects regression models accounting for study and site heterogeneity were used to explore factors affecting PC1/2 and risk of recrudescence within areas with reported delayed parasite clearance (western Cambodia, western Thailand after 2000, southern Vietnam, southern Myanmar) and in all other areas where parasite populations are artemisinin sensitive. Results PC1/2 was estimated in 6975 patients, 3288 of whom also had treatment outcomes evaluate d during 28–63 days follow-up, with 93 (2.8 %) PCR-confirmed recrudescences. In areas with artemisinin-sensitive parasites, the median PC1/2 following three-day artesunate treatment (4 mg/kg/day) ranged from 1.8 to 3.0 h and the proportion of patients with PC1/2 >5 h from 0 to 10 %. Artesunate doses of 4 mg/kg/day decreased PC1/2 by 8.1 % (95 % CI 3.2–12.6) compared to 2 mg/kg/day, except in populations with delayed parasite clearance. PC1/2 was longer in children and in patients with fever or anaemia at enrolment. Long PC1/2 (HR = 2.91, 95 % CI 1.95–4.34 for twofold increase, p

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.

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., Author Summary Plasmodium vivax causes tens of millions of malaria cases each year. Although some vaccines against P. vivax are being developed, little is known about the geospatial genetic diversity and selective constraints of the parasite surface antigens that these vaccines target. In order to create vaccines that are both efficacious and useful in diverse regions of the world, the strain diversity of these potential vaccine targets must be well understood. Specifically, we must understand whether and how the human immune system develops immunity against these antigens as well as understanding whether these antigens are similar in geographically diverse parasite populations. Here, using next-generation sequencing and population-genetic analyses, we found evidence of likely immune selection in specific regions of two leading vivax vaccine candidate antigens, PvMSP-1 and PvCSP. At the pvmsp-1 locus, we also found more genetic variability within populations than between populations, with some DNA sequences from geographically diverse populations being highly similar. In contrast, pvcsp sequences from geographically diverse populations are very distinct from one another, with specific sequence patterns occurring in certain geographic regions. Our findings provide new insights into the geographic genetic diversity of these two antigens and can help inform the development of effective P. vivax vaccines.

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.

Background The emergence of artemisinin resistance in South East Asia threatens the efficacy of artemisinin derivatives (AD). Since the pharmacodynamic hallmark of AD is rapid parasite clearance, the clinical phenotype of slow clearance characterises resistance. This indicator remains critically important to monitor the extent of the problem in the absence of molecular marker(s) associated with artemisinin resistance and lack of sensitivity of current in vitro tests. Frequent parasite counts are needed to define clearance rate but it is uncertain what sampling frequency is required to ensure reliable estimates.

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 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.

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.

Whilst some populations have recently experienced dramatic declines in malaria, the majority of those most at risk of Plasmodium falciparum malaria still lack access to effective treatment with artemisinin combination therapy (ACT) and others are already facing parasites resistant to artemisinins. In this context, there is a crucial need to improve both access to and targeting of ACT through greater availability of good quality ACT and parasitological diagnosis. This is an issue of increasing urgency notably in the private commercial sector, which, in many countries, plays an important role in the provision of malaria treatment. The Affordable Medicines Facility for malaria (AMFm) is a recent initiative that aims to increase the provision of affordable ACT in public, private and NGO sectors through a manufacturer-level subsidy. However, to date, there is little documented experience in the programmatic implementation of subsidized ACT in the private sector. Cambodia is in the unique position of having more than 10 years of experience not only in implementing subsidized ACT, but also rapid diagnostic tests (RDT) as part of a nationwide social marketing programme. The programme includes behaviour change communication and the training of private providers as well as the sale and distribution of Malarine, the recommended ACT, and Malacheck, the RDT. This paper describes and evaluates this experience by drawing on the results of household and provider surveys conducted since the start of the programme. The available evidence suggests that providers' and consumers' awareness of Malarine increased rapidly, but that of Malacheck much less so. In addition, improvements in ACT and RDT availability and uptake were relatively slow, particularly in more remote areas. The lack of standardization in the survey methods and the gaps in the data highlight the importance of establishing a clear system for monitoring and evaluation for similar initiatives. Despite these limitations, a number of important lessons can still be learnt. These include the importance of a comprehensive communications strategy and of a sustained and reliable supply of products, with attention to the geographical reach of both. Other important challenges relate to the difficulty in incentivising providers and consumers not only to choose the recommended drug, but to precede this with a confirmatory blood test and ensure that providers adhere to the test results and patients to the treatment regime. In Cambodia, this is particularly complicated due to problems inherent to the drug itself and the emergence of artemisinin resistance.

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.

Fecal examinations using the Kato Katz technique were performed on a total of 1,287 villagers (945 students and 342 general inhabitants) of Oddar Meanchey Province, Cambodia in May 2007 and November 2009. The overall intestinal helminth egg positive rate was 23.9%, and the most prevalent helminth species was hookworms (21.6%). Other helminth eggs detected included echinostomes (1.0%), Enterobius vermicularis (0.8%), small trematode eggs (0.7%), which may include Opisthorchis viverrini and Haplorchis spp., and Hymenolepis nana (0.4%). In order to recover adult echinostomes, we treated 2 patients with 10-15 mg/kg praziquantel and purged. Total 14 adult echinostomes, 1 and 13 worms from each patient, were collected. The echinostomes characteristically had 49-51 collar spines and 2 round or slightly lobated testes. They were identified as Echinostoma ilocanum (Garrison, 1908) Odhner, 1911. So far as literature are concerned, this is the first record on the discovery of human E. ilocanum infection in Cambodia.

Background Drug resistance of falciparum malaria is a global problem. Sulphadoxine/pyrimethamine-resistant and mefloquine-resistant strains of falciparum malaria have spread in Southeast Asia at lightning speed in 1980s-1990s, and the Cambodia-Thailand border is one of the malaria epidemic areas with the most severe forms of multi-drug resistant falciparum malaria. Methods Artemisinin-piperaquine (AP), dihydroartemisinin-piperaquine phosphate (DHP) and artemether-lumefantrine (AL) were used to treat 110, 55 and 55 uncomplicated malaria patients, respectively. The total dosage for adults is 1,750 mg (four tablets, twice over 24 hours) of AP, 2,880 mg (eight tablets, four times over two days) of DHP, and 3,360 mg (24 tablets, six times over three days) of AL. The 28-day cure rate, parasite clearance time, fever clearance time, and drug tolerance of patients to the three drugs were compared. All of the above methods were consistent with the current national guidelines. Results The mean parasite clearance time was similar in all three groups (66.7 ± 21.9 hrs, 65.6 ± 27.3 hrs, 65.3 ± 22.5 hrs in AP, DHP and AL groups, respectively), and there was no remarkable difference between them; the fever clearance time was also similar (31.6 ± 17.7 hrs, 34.6 ± 21.8 hrs and 36.9 ± 15.4 hrs, respectively). After following up for 28-days, the cure rate was 95.1%(97/102), 98.2%(54/55) and 82.4%(42/51); and the recrudescence cases was 4.9%(5/102), 1.8%(1/55) and 17.6%(9/51), respectively. Therefore, the statistical data showed that 28-day cure rate in AP and DHP groups was superior to AL group obviously. The patients had good tolerance to all the three drugs, and some side effects (anoxia, nausea, vomiting, headache and dizziness) could be found in every group and they were self-limited; patients in control groups also had good tolerance to DHP and AL, there was no remarkable difference in the three groups. Conclusions AP, DHP and AL all remained efficacious treatments for the treatment of falciparum malaria in Cambodia-Thailand border area. However, in this particular setting, the AP regimen turned out to be favourable in terms of efficacy and effectiveness, simplicity of administration, cost and compliance. Trial Registration The trial was registered at Chinese Clinical Trial Register under identifier 2005L01041.

Background The emergence of artemisinin resistance has raised concerns that the most potent antimalarial drug may be under threat. The currently recommended daily dose of artesunate (AS) is 4 mg/kg, and is administered for 3 days together with a partner antimalarial drug. This study investigated the impact of different AS doses on clinical and parasitological responses in malaria patients from an area of known artemisinin resistance in western Cambodia. Methods Adult patients with uncomplicated P. falciparum malaria were randomized into one of three 7-day AS monotherapy regimens: 2, 4 or 6 mg/kg/day (total dose 14, 28 and 42 mg/kg). Clinical, parasitological, pharmacokinetic and in vitro drug sensitivity data was collected over a 7-day inpatient period and during weekly follow-up to 42 days. Results 143 patients were enrolled (n = 75, 40 and 28 to receive AS 2, 4 and 6 mg/kg/day respectively). Cure rates were high in all treatment groups at 42 days despite almost half the patients remaining parasitemic on Day 3. There was no impact of increasing AS dose on median parasite clearance times, median parasite clearance rates or on the proportion of patients remaining parasitemic on Day 3. However at the lowest dose used (2 mg/kg/d) patients with parasitemia >10,000/µL had longer median (IQR) parasite clearance times than those with parasitemia

The diversity in the Plasmodium falciparum genome can be used to explore parasite population dynamics, with practical applications to malaria control. The ability to identify the geographic origin and trace the migratory patterns of parasites with clinically important phenotypes such as drug resistance is particularly relevant. With increasing single-nucleotide polymorphism (SNP) discovery from ongoing Plasmodium genome sequencing projects, a demand for high SNP and sample throughput genotyping platforms for large-scale population genetic studies is required. Low parasitaemias and multiple clone infections present a number of challenges to genotyping P. falciparum. We addressed some of these issues using a custom 384-SNP Illumina GoldenGate assay on P. falciparum DNA from laboratory clones (long-term cultured adapted parasite clones), short-term cultured parasite isolates and clinical (non-cultured isolates) samples from East and West Africa, Southeast Asia and Oceania. Eighty percent of the SNPs (n = 306) produced reliable genotype calls on samples containing as little as 2 ng of total genomic DNA and on whole genome amplified DNA. Analysis of artificial mixtures of laboratory clones demonstrated high genotype calling specificity and moderate sensitivity to call minor frequency alleles. Clear resolution of geographically distinct populations was demonstrated using Principal Components Analysis (PCA), and global patterns of population genetic diversity were consistent with previous reports. These results validate the utility of the platform in performing population genetic studies of P. falciparum.

BACKGROUND: Fears of emerging artemisinin resistance in western Cambodia have prompted a series of clinical trials investigating whether slow responses to antimalarial treatment can be overcome by increasing doses of drug. METHODS: Patients with uncomplicated malaria were allocated 1 of 3 oral artesunate monotherapy regimens (2, 4, or 6 mg/kg/day for 7 days) and were observed for 42 days. A series of safety measures, including complete blood count on days 0, 3, 6, and 14, was implemented because of a lack of safety data for these experimental doses. RESULTS: After 3 doses, geometric mean absolute neutrophil counts were reduced in all groups, and 2 patients required artesunate to be discontinued because of neutropenia (absolute neutrophil count