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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.

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.

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.

In efforts to reduce the global burden of soil transmitted helminth (STH) infections in school age children (SAC, 6-14 years old), Children Without Worms donates mebendazole to 8 countries with high prevalence of STH infections. Cambodia's national deworming program currently targets SAC through bi-annual school-based distributions of a single dose of mebendazole. A 30-cluster household survey was conducted in the rural province Kampot, to validate mebendazole treatment coverage in SAC and to assess the level of improved water supply and sanitation. Bi-annual primary school-based distributions proved to be an effective strategy in reaching school attending SAC, with treatment coverage rates between 84.1% and 88.8%. However, significantly lower rates (23.3-48.8%) were seen among SAC not enrolled in primary schools. Often members of the most marginalized families of the community, they are particularly at risk of STH infection. Methods to reach these children need to be explored to avoid treatment inequities.

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.

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.

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.

In the Mekong River basin, the first case of schistosomiasis was reported in 1957. In the 1960s, endemic areas of the infection, of which profiles were similar to those of schistosomiasis japonica, were discovered in Khong Island, Laos, to Kratie province, Cambodia. A new intermediate snail host; Neotricula aperta was identified and the Mekong strain of schistosome was elevated to a new species: Schistosoma mekongi in 1978. Baseline epidemiological surveillance was performed and schistosomiasis mekongi was described as a public health implication in the middle Mekong River basin. Because of political and economical confusion, endemic situation had become worse, and no control program had been implemented until mass treatment program with praziquantel on Khong Island in 1983. Since then, the prevalence of S. mekongi infection has rapidly decreased in each endemic area. Serological diagnosis has been useful to detect new but low endemic foci. Clinical manifestations of S. mekongi infection are similar to those of S. mansoni and S. japonicum infections. As the reduction of prevalence and intensity of S. mekongi infection, morbidity due to the disease has changed, and ultrasonographic examination is now useful to evaluate morbidity due to schistosomiasis mekongi. Transmission of the disease occurs in a couple of months during low water season. Control of N. aperta is difficult and long-lasting effective control measurements have, so far, not been available. In the next step for controling S. mekongi infection, mass treatment should be continued, and it is needed to combine other appropriate control activities.

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.

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.

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.

Schistosomiasis mekongi is an important public health issue in endemic countries. In this study, we evaluated an indirect immunodiagnostic ELISA method using Schistosoma mekongi soluble egg antigen. Sodium metaperiodate (SMP)-ELISA was utilized in order to remove the glycosylated epitopes responsible for false positive reactions and the results using this method were compared with those using conventional ELISA (conv-ELISA). Forty-two serum samples from schistosomiasis mekongi egg-positive patients and 100 serum samples from schistosomiasis-negative Cambodian subjects were tested using both ELISA methods. The ranges of ELISA values for positive and negative sera were distinct on SMP-ELISA, but the ranges of the two groups of sera overlapped on conv-ELISA. Therefore, diagnostic criteria may be established based on the highest ELISA value on negative sera and the lowest ELISA value on positive sera. In the present study, both the sensitivity and specificity of SMP-ELISA reached 100% using the criteria in which an ELISA valueor = 0.2 was positive.

To determine the prevalence of helminthic infections in Pursat Province, Cambodia, we tested fecal specimens from 471 children, 10–14 years of age, in June 2007. The prevalence of infection with echinostome flukes ranged from 7.5% to 22.4% in 4 schools surveyed. Adult worms were identified as Echinostoma revolutum.

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

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

Background Increasing rates of failure of artemisinin-based combination therapy have highlighted the possibility of emerging artemisinin resistance along the Thai-Cambodian border. We used an integrated in vivo-in vitro approach to assess the presence of artemisinin resistance in western Cambodia. This article provides additional data from a clinical trial that has been published in The New England Journal of Medicine. Methods Ninety-four adult patients from Battambang Province, western Cambodia, who presented with uncomplicated falciparum malaria were randomized to receive high-dose artesunate therapy (4 mg/kg/day orally for 7 days) or quinine-tetracycline. Plasma concentrations of dihydroartemisinin, in vitro drug susceptibility, and molecular markers were analyzed. Cases meeting all the following criteria were classified as artemisinin resistant: failure to clear parasites within 7 days of treatment or reemergence of parasites within 28 days of follow-up; adequate plasma concentrations of dihydroartemisinin; prolonged parasite clearance; and increased in vitro drug susceptibility levels for dihydroartemisinin. Results Two (3.3%) of 60 artesunate-treated patients were classified as artemisinin resistant. Their parasite clearance times were prolonged (133 and 95 h, compared with a median of 52.2 h in patients who were cured). These patients had 50% inhibitory concentrations of dihydroartemisinin that were almost 10 times higher than the reference clone W2. Resistance did not appear to be mediated by the pfmdr1 copy number or selected PfATPase6 polymorphisms previously proposed to confer artemisinin resistance. Conclusion Artemisinin resistance has emerged along the Thai-Cambodian border. The potentially devastating implications of spreading resistance to a drug that currently has no successor call for further studies of this emerging problem. Clinical trial registration ClinicalTrials.gov identifier NCT00479206.

Artemisinin combination therapies are the first-line treatments for uncomplicated Plasmodium falciparum malaria in most malaria-endemic countries. Recently, partial artemisinin-resistant P. falciparum malaria has emerged on the Cambodia-Thailand border. Exposure of the parasite population to artemisinin monotherapies in subtherapeutic doses for over 30 years, and the availability of substandard artemisinins, have probably been the main driving force in the selection of the resistant phenotype in the region. A multifaceted containment programme has recently been launched, including early diagnosis and appropriate treatment, decreasing drug pressure, optimising vector control, targeting the mobile population, strengthening management and surveillance systems, and operational research. Mathematical modelling can be a useful tool to evaluate possible strategies for containment.

Background With the increase in use of point-of-care diagnostic tests for malaria and other diseases comes the necessity of storing the diagnostic kits and the drugs required for subsequent management, in remote areas, where temperatures are high and electricity supply is unreliable or unavailable. Methods To address the lack of temperature-controlled storage during the introduction of community-based malaria management in Cambodia, the Cambodian National Centre for Parasitology, Entomology and Malaria Control (CNM) developed prototype evaporative cooling boxes (Cambodian Cooler Boxes - CCBs) for storage of perishable medical commodities in remote clinics. The performance of these CCBs for maintaining suitable storage temperatures was evaluated over two phases in 2005 and 2006-7, comparing conditions in CCBs using water as designed, CCBs with no water for evaporation, and ambient storage room temperatures. Temperature and humidity was monitored, together with the capacity of the RDTs recommended for storage between 2 to 30 degree Celsius to detect low-density malaria parasite samples after storage under these conditions. Results Significant differences were recorded between the proportion of temperatures within the recommended RDT storage conditions in the CCBs with water and the temperatures in the storage room (p < 0.001) and maximum temperatures were lower. RDTs stored at ambient temperatures were negative when tested with parasitized blood (2,000 parasites per micro litre) at 210 days, while the field RDTs kept in CCBs with water gave positive results until 360 days. Discussion and Conclusions The CCB was an effective tool for storage of RDTs at optimal conditions, and extended the effective life-span of the tests. The concept of evaporative cooling has potential to greatly enhance access to perishable diagnostics and medicines in remote communities, as it allows prolonged storage at low cost using locally-available materials, in the absence of electricity.

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.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.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.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.

Background Resistance to anti-malarial drugs hampers control efforts and increases the risk of morbidity and mortality from malaria. The efficacy of standard therapies for uncomplicated Plasmodium falciparum and Plasmodium vivax malaria was assessed in Chumkiri, Kampot Province, Cambodia. Methods One hundred fifty-one subjects with uncomplicated falciparum malaria received directly observed therapy with 12 mg/kg artesunate (over three days) and 25 mg/kg mefloquine, up to a maximum dose of 600 mg artesunate/1,000 mg mefloquine. One hundred nine subjects with uncomplicated vivax malaria received a total of 25 mg/kg chloroquine, up to a maximum dose of 1,500 mg, over three days. Subjects were followed for 42 days or until recurrent parasitaemia was observed. For P. falciparum infected subjects, PCR genotyping of msp1, msp2, and glurp was used to distinguish treatment failures from new infections. Treatment failure rates at days 28 and 42 were analyzed using both per protocol and Kaplan-Meier survival analysis. Real Time PCR was used to measure the copy number of the pfmdr1 gene and standard 48-hour isotopic hypoxanthine incorporation assays were used to measure IC50 for anti-malarial drugs. Results Among P. falciparum infected subjects, 47.0% were still parasitemic on day 2 and 11.3% on day 3. The PCR corrected treatment failure rates determined by survival analysis at 28 and 42 days were 13.1% and 18.8%, respectively. Treatment failure was associated with increased pfmdr1 copy number, higher initial parasitaemia, higher mefloquine IC50, and longer time to parasite clearance. One P. falciparum isolate, from a treatment failure, had markedly elevated IC50 for both mefloquine (130 nM) and artesunate (6.7 nM). Among P. vivax infected subjects, 42.1% suffered recurrent P. vivax parasitaemia. None acquired new P. falciparum infection. Conclusion The results suggest that artesunate-mefloquine combination therapy is beginning to fail in southern Cambodia and that resistance is not confined to the provinces at the Thai-Cambodian border. It is unclear whether the treatment failures are due solely to mefloquine resistance or to artesunate resistance as well. The findings of delayed clearance times and elevated artesunate IC50 suggest that artesunate resistance may be emerging on a background of mefloquine resistance.

A field evaluation of a novel, 5% controlled-release formulation of pyriproxyfen was carried out in 400-500 liter concrete water storage jars, the most common and important larval habitat of the dengue vector, Aedes aegypti, in Cambodia. The formulation consisted of cylindrical resin strands, 3 mm in diameter and 40 mm in length. Pyriproxyfen was applied to 100 jars at a target dose of 0.03-0.04 mg of active ingredient (a.i.) per liter (30-40 ppb) in households in Phum Thmei, a village near the capital city of Phnom Penh, in April 2005. Inhibition of adult emergence (IE) in field populations ofAe. aegypti was measured every 2 weeks for 34 weeks. IE in treated jars exceeded 90% for 20 weeks, and remained above 80% until the end of the study. In 25 untreated jars, failure of pupae to metamorphose into viable adults remained below 4%. No alteration of taste or other undesirable effects of the treatment were reported by householders. This single treatment provided control of Ae. aegypti in water jars for the length of the main dengue transmission season in Cambodia that normally extends from May to November.

Background Malaria-endemic countries are switching antimalarial drug policy from cheap ineffective monotherapies to artemisinin combination therapies (ACTs) for the treatment of Plasmodium falciparum malaria and the global community are considering setting up a global subsidy to fund their purchase. However, in order to ensure that ACTs are correctly used and are accessible to the poor and remote communities who need them, specific interventions will be necessary and the additional costs need to be considered. Methods This paper presents an incremental cost analysis of some of these interventions in Cambodia, the first country to change national antimalarial drug policy to an ACT of artesunate and mefloquine. These costs include the cost of rapid diagnostic tests (RDTs), the cost of blister-packaging the drugs locally and the costs of increasing access to diagnosis and treatment to remote communities through malaria outreach teams (MOTs) and Village Malaria Workers (VMW). Results At optimum productive capacity, the cost of blister-packaging cost under $0.20 per package but in reality was significantly more than this because of the low rate of production. The annual fixed cost (exclusive of RDTs and drugs) per capita of the MOT and VMW schemes was $0.44 and $0.69 respectively. However because the VMW scheme achieved a higher rate of coverage than the MOT scheme, the cost per patient treated was substantially lower at $5.14 compared to $12.74 per falciparum malaria patient treated. The annual cost inclusive of the RDTs and drugs was $19.31 for the MOT scheme and $11.28 for the VMW scheme given similar RDT positivity rates of around 22% and good provider compliance to test results. Conclusion In addition to the cost of ACTs themselves, substantial additional investments are required in order to ensure that they reach the targeted population via appropriate delivery systems and to ensure that they are used appropriately. In addition, differences in local conditions, in particular the prevalence of malaria and the pre-existing infrastructure, need to be considered in choosing appropriate diagnostic and delivery strategies.

We constructed a mathematical model for the transmission of Schistosoma mekongi in Cambodia. The simulation of the model will be instrumental in planning schistosomiasis control measures. The model includes two definitive hosts, humans and dogs, as animal reservoirs. Dogs are recognized to play an important role in schistosomiasis transmission in Cambodia. For the purpose of dealing with age-specific prevalence and intensity of infection, the human population was classified into eight age categories in the model. To describe the seasonal fluctuation of the intermediate host population of S. mekongi, the "Post-Spate Survival" hypothesis was adopted for the population dynamics of Neotricula aperta present in the Mekong River. We carried out simulations to evaluate the effect of universal treatment (UT) and targeted mass treatment (TT) with praziquantel on the reduction in prevalence of S. mekongi. The simulations indicated that biyearly UT for 8 years or yearly TT for 5 years after three courses of yearly UT could reduce the prevalence to below 5% when a UT or TT coverage of 85% of inhabitants was achieved. The simulation suggested that the suppression of S. mekongi in Cambodia would be possible by UT or TT with a high coverage rate.

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.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.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.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.

The objective of this study was to study Schistosoma mekongi and other intestinal parasitic infections, and intestinal symptoms and related complaints among school-age children and adolescents living around Tonle Sap Lake. Villages were selected where there were potential signs of schistosomiasis (hepatomegaly), and where subjects complained of intestinal symptoms. Stool samples were collected from 1,616 children and were examined by Kato-Katz, SAF concentration, and Baermann technique; short clinical examinations were also performed. No S. mekongi infection was detected, although a high level of intense human water contacts was reported. Helminth infection such as Ascaris lumbricoides (27.7%) and hookworms (29.7%) were common. Trichuris trichiura 4.4%), Hymenolepis nana (6.2%), Giardia lamblia (4.2%), and Entamoeba spp (14.4%) were also recorded. Strongyloides stercoralis was frequently diagnosed (20.2%). It was concluded that it is unlikely that S. mekongi is transmitted in Tonle Sap Lake. However, other intestinal parasitic infections are widespread. In particular, S. stercoralis should be considered an important etiologic agent in children and adolescents with abdominal complaints.

Artesunate and mefloquine combination treatment has been used since 2000 in Cambodia as the first-line drug for the treatment of uncomplicated falciparum malaria. In order to assess its efficacy and safety, the national malaria control programme conducted 14 therapeutic efficacy studies with the drug combination between 2001 and 2004 at nine sites. In 2001 and 2002, co-blister packs of artesunate and mefloquine were used, whereas in 2003 and 2004, drugs were given individually from a bulk pack at a total dose of 12 mg/kg of artesunate and 25 mg/kg of mefloquine over 3 days. A total of 1025 patients were enrolled over the 4 years and 977 were follow-up during the period of 28 days. The PCR-corrected cure rates ranged from 85.7% to 100% with an overall cure rate of 95.8% (920/960). The studies in 2002 showed also that co-blister packs used on the basis of age and not on the basis of weight could lead to underdosed regimens but without any detectable effect on the treatment outcome. The follow-up period was extended from 28 to 42 days in three sites in 2004. A total of 219 among 255 were follow-up until day 42. The cure rate decreased but not significantly from 90.1% (73/81) with 28 days follow-up to 79.3% (46/58) with 42 days follow-up in Pailin, whereas the cure rate remained at 100% in the two other sites. Side effects were common, especially dizziness, but were mild and transient and patients recovered without any medical intervention.

Concrete domestic water-storage jars are a common larval habitat of the dengue vector Aedes aegypti in countries of Southeast Asia. The efficacy of a novel controlled-release formulation of the insect growth regulator pyriproxyfen, designed to inhibit adult emergence for 6 months (the approximate duration of the main dengue transmission season in many endemic countries) was tested in Cambodia against a local strain of Ae. aegypti in 200-liter jars. The resin-based formulation contained 4.8% active ingredient (AI). At target dosages of 18, 27, and 36 ppb of AI, inhibition of adult emergence remained above 95% for at least 2 months. After 3 months at 18 ppb AI, the residual efficacy was significantly lower than for the higher dosages (P0.05). At the higher dosages, inhibition of adult emergence wasor = 87% for 6 months. At a dosage of 27 ppb AI, monthly removal and replacement of two thirds of the water did not reduce efficacy (P0.05). Potential operational advantages and challenges of using the formulation are discussed. Larger-scale efficacy studies are recommended in community settings.

Schistosomiasis mekongi is endemic in the Mekong River basin; about 80,000 people are at risk of infection in Cambodia. We conducted ultrasonographic studies of patients with schistosomiasis mekongi in Kratie province, Cambodia, focusing especially on the relationship between the frequency of praziquantel treatment and findings of ultrasonographic imaging. The frequency of praziquantel treatment in the period from 1995 to 2002 was classified into four groups: 1-2, 3-4, 5-6, and 7-8 times. Ultrasonographic images were examined to determine the presence of thickening of the portal vein wall and formation of meandering collateral circulation of the splenic vein. We selected these parameters because they are unaffected by interobserver bias. The results showed that thickening of the portal vein wall may have potential to improve with frequent praziquantel treatment. On the other hand, established hard splenomegaly and meandering collateral circulation of the splenic vein, improved very little with praziquantel treatment.

Counterfeit and substandard antimalarial drugs can cause death and contribute to the growing malaria drug resistance problem, particularly in Southeast Asia. Since 2003 in Cambodia the quality of antimalarial drugs both in the public and private health sector is regularly monitored in sentinel sites. We surveyed 34% of all 498 known facilities and drug outlets in four provinces. We collected 451 drug samples; 79% of these were not registered at the Cambodia Department of Drugs and Food (DDF). Twenty-seven percent of the samples failed the thin layer chromatography and disintegration tests; all of them were unregistered products. Immediate action against counterfeit drugs was taken by the National Malaria Control Program (NMCP) and the DDF. They communicated with the Provincial Health Department about the presence of counterfeit antimalarial drugs through alert letters, a manual, annual malaria conferencing and other training occasions. Television campaigns to alert the population about counterfeit drugs were conducted. Moreover, the NMCP has been promoting the use of good quality antimalarial drugs of a blister co-packaged combination of artesunate and mefloquine in public and private sectors. Appropriate strategies need to be developed and implemented by relevant government agencies and stakeholders to strengthen drug quality assurance and control systems in the country.

We conducted a survey of malaria diagnoses and glucose-6-phosphate dehydrogenase (G6PD) testing in remote areas of Cambodia. Blood specimens from 670 people were collected by the finger-prick method. Of these people, 24.9% were found to have malaria, and 7.0% of people were G6PD deficient. In the Khmer, the largest ethnical population in Cambodia, the G6PD deficiency rate of males was 12.6% (25/199) whereas the rates in the minorities of the Tum Pun and the Cha Ray were 1.1% (1/93) and 3.2% (2/63), respectively. Of the G6PD-deficient subjects, 97.9% (46/47) were G6PD Viangchan (871G>A), and only one case (2.1%) was G6PD Union (1360C>T). Since G6PD Mahidol (487G>A) is common in Myanmar according to our previous study, the current finding suggests that the Cambodian population is derived from homogeneous ancestries and is different from the Myanmar population. All G6PD Viangchan cases were linked to two other mutations of 1311C>T and IVS-11 nt93T>C in the G6PD gene.

We have been conducting surveys of schistosomiasis mekongi along the Mekong river in Cambodia since 1997. We attempted to detect canine schistosome infection during the survey in 2000 because dogs were reported to be natural reservoirs of the Mekong schistosome in Lao PDR. A total of 28 canine fecal samples were collected in Kbal Chuor village, Kratie Province and examined for schistosome eggs. One specimen had schistosome eggs (positive rate = 3.6%; egg density = 100/gram stool), which showed characteristics of Schistosoma mekongi. During the 2001 survey, one out of 310 canine stool samples was positive for schistosome eggs (positive rate = 0.32%; egg density = 3,456/gram stool). These are the first confirmed cases of canine schistosomiasis mekongi in Cambodia, which suggests that dogs are animal reservoirs of S. mekongi in the survey area. We further tried to detect S. mekongi in cows, water buffalos, pigs,horses, and field rats in five villages in Kratie Province; no schistosome egg was found in the stools of these animals.

To the Editor: Although artemisinins are potent and rapidly acting antimalarial drugs, their widespread use for treating patients with Plasmodium falciparum malaria raises the question of emerging drug resistance.1,2 Artemisinin monotherapy should not be used in areas where malaria is endemic; it requires an extended administration period and may lead to treatment failure, most frequently because of problems with compliance. Recent reports of high failure rates associated with artemisinin-based combination therapy, as well as in vitro drug-susceptibility data, suggest the possibility of clinical artemisinin resistance along the Thai–Cambodian border.3,4 We studied the potential emergence of artemisinin resistance using . . .

We collected fecal samples from 21 individuals infected with Taenia tapeworms in Koh Kong Province, Cambodia, and performed nucleotide sequencing of the cox1 gene and multiplex PCR on the eggs for DNA differential diagnosis of human Taenia tapeworms. Genomic DNA was extracted from the eggs of a minimum number of 10 isolated from fecal samples. Using oligonucleotide primers Ta7126F, Ts7313F, Tso7466F, and Rev7915, the multiplex PCR assay proved useful for differentially diagnosing Taenia solium, Taenia saginata, and Taenia asiatica based on 706, 629, and 474 bp bands, respectively. All of the Taenia specimens from Kho Kong, Cambodia, were identified as either T. saginata (n=19) or T. solium (n=2) by cox1 sequencing and multiplex PCR.

Background Malaria microscopy and rapid diagnostic tests are insensitive for very low-density parasitaemia. This insensitivity may lead to missed asymptomatic sub-microscopic parasitaemia, a potential reservoir for infection. Similarly, mixed infections and interactions between Plasmodium species may be missed. The objectives were first to develop a rapid and sensitive PCR-based diagnostic method to detect low parasitaemia and mixed infections, and then to investigate the epidemiological importance of sub-microscopic and mixed infections in Rattanakiri Province, Cambodia. Methods A new malaria diagnostic method, using restriction fragment length polymorphism analysis of the cytochrome b genes of the four human Plasmodium species and denaturing high performance liquid chromatography, has been developed. The results of this RFLP-dHPLC method have been compared to 1) traditional nested PCR amplification of the 18S rRNA gene, 2) sequencing of the amplified fragments of the cytochrome b gene and 3) microscopy. Blood spots on filter paper and Giemsa-stained blood thick smears collected in 2001 from 1,356 inhabitants of eight villages of Rattanakiri Province have been analysed by the RFLP-dHPLC method and microscopy to assess the prevalence of sub-microscopic and mixed infections. Results The sensitivity and specificity of the new RFLP-dHPLC was similar to that of the other molecular methods. The RFLP-dHPLC method was more sensitive and specific than microscopy, particularly for detecting low-level parasitaemia and mixed infections. In Rattanakiri Province, the prevalences of Plasmodium falciparum and Plasmodium vivax were approximately two-fold and three-fold higher, respectively, by RFLP-dHPLC (59% and 15%, respectively) than by microscopy (28% and 5%, respectively). In addition, Plasmodium ovale and Plasmodium malariae were never detected by microscopy, while they were detected by RFLP-dHPLC, in 11.2% and 1.3% of the blood samples, respectively. Moreover, the proportion of mixed infections detected by RFLP-dHPLC was higher (23%) than with microscopy (8%). Conclusions The rapid and sensitive molecular diagnosis method developed here could be considered for mass screening and ACT treatment of inhabitants of low-endemicity areas of Southeast Asia.