Major pitfalls in the measurement of artemisinin derivatives in plasma in clinical studies.

A bioanalytical method for the analysis of artesunate (ARS) and its metabolite dihydroartemisinin (DHA) in human plasma using protein precipitation and liquid chromatography coupled to positive tandem mass spectroscopy was developed. The method was validated according to published US FDA-guidelines and showed excellent performance. However, when it was applied to clinical pharmacokinetic studies in malaria, variable degradation of the artemisinins introduced an unacceptable large source of error, rendering the assay useless. Haemolytic products related to sample collection and malaria infection degraded the compounds. Addition of organic solvents during sample processing and even low volume addition of the internal standard in an organic solvent caused degradation. A solid phase extraction method avoiding organic solvents eliminated problems arising from haemolysis induced degradation. Plasma esterases mediated only approximately 20% of ex vivo hydrolysis of ARS into DHA. There are multiple sources of major preventable error in measuring ARS and DHA in plasma samples from clinical trials. These various pitfalls have undoubtedly contributed to the large inter-subject variation in plasma concentration profiles and derived pharmacokinetic parameters for these important antimalarial drugs.

Prevention of lymphatic filariasis with insecticide-treated bednets in Cambodia.

Potential risk factors for lymphatic filariasis (LF), including the failure to use insecticide-treated bednets (ITN), were studied in four north-eastern provinces of Cambodia, using 43 cases of LF and 248 apparently healthy controls who were matched with the cases in terms of age (+/-5 years), gender and village. The results of a univariate matched analysis indicated that lack of ITN [odds ratio (OR)=20.1; 95% confidence interval (CI)=2.2-182.4; P=0.008] and frequent overnight stays in forests or paddy fields outside the village (OR=3.6; CI=1.3-10.0; P=0.012) were the most important risk factors. In a multivariate matched analysis, frequent overnight stays outside the village combined with bednet use showed a clear dose-response relationship, with untreated nets offering significant protection during such stays but ITN offering significantly better protection. Even if villagers used ITN during such stays, they were significantly more likely to develop LF than villagers who did not travel (OR=5.0; CI=1.5-16.3; P=0.008), and if they used untreated or no nets while away they were even more likely than the non-travellers to develop the disease (OR=1413; CI=28.0-71,189; P<0.001). Taken together, these results provide further evidence for the importance of bednet use and their impregnation in the prevention of LF. At least in north-eastern Cambodia, the use of bednets and, preferably, ITN is particularly important during overnight stays outside the home village. In areas where LF is endemic, the sustained distribution of insecticide-treated bednets or hammock nets appears to be a very good idea.

A randomized open study to assess the efficacy and tolerability of dihydroartemisinin-piperaquine for the treatment of uncomplicated falciparum malaria in Cambodia.

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.

Control of Schistosoma mekongi in Cambodia: results of eight years of control activities in the two endemic provinces.

In Cambodia, schistosomiasis is transmitted in the provinces of Kratie and Stung Treng where approximately 80000 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 (40mg/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 (500mg) 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.

Insecticide-treated bednets for the prevention of Plasmodium falciparum malaria in Cambodia: a cluster-randomized trial.

OBJECTIVES: To validate and quantify the impact of insecticide-treated bednets (ITN) on malaria morbidity and mortality in Cambodia. METHODS: A paired, cluster-randomized trial of ITN was conducted in Rattanakiri, North East Cambodia. Thirty-four villages with a total population of 10,726 were randomized to receive deltamethrin-impregnated bednets or to control (no net provision). Cross-sectional surveys measured Plasmodium falciparum prevalence at baseline and 10 months after ITN distribution. Village malaria volunteers in control and intervention villages treated dipstick-positive P. falciparum cases with artesunate and mefloquine. The resulting passive surveillance data were used as an estimate of the incidence of clinical P. falciparum infections. RESULTS: There was a protective efficacy of 28% in P. falciparum incidence (adjusted rate ratio 0.72, 95% CI 0.47-1.08) and 9% in P. falciparum prevalence (adjusted prevalence ratio 0.91, 95% CI 0.65-1.28) in ITN relative to control villages; however, neither of these estimates reached statistical significance. Individual-level analysis indicated a greater reduction in P. falciparum prevalence among under 5-year-olds (adjusted OR = 0.63, 95% CI 0.26-1.53) compared to older individuals (interaction P = 0.042). The protective efficacy of 35% (95% CI -28, 67%) with respect to clinical P. falciparum incidence in under 5-year-olds was more pronounced than the corresponding estimates for prevalence but was again not significant. CONCLUSIONS: Lack of statistical significance in the results is likely to be due to a lack of power. The analysis provides further evidence for ITN effectiveness in South East Asia, particularly among individuals under 5 years of age.

Counterfeit and substandard antimalarial drugs in Cambodia.

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.

[Place of surgery in the prevention of recurrences of digestive haemorrhages at the patients presenting a portal hypertension due to Schistosoma mekongi]. / Place de la chirurgie dans la prévention des récidives d'hémorragies digestives chez les sujets présentant une hypertension portale due a Schistosoma mekongi.

In spite of a decrease of the prevalence of hepato-splenic schistosomiasis thanks to mass-treatment with Praziquentel from December 1994 till now (CNM - MSF - WHO - Health Provincial Director) of target-populations in Kratie Province, severe cases of portal hypertension are not exceptional (digestive bleedings, after rupture of oesophageal varices). Out of 106 cases of portal hypertension: alI patients have had clinical survey biological tests (liver function, haematology and serology). Most of them had ultrasonography (Aloka 55,500 Sound 3.5 MHz). Nearly half of the group of 153 patients has never had bleedings. More than 45 were not eligible for surgery for different reasons: severe anaemia (few possibilities for massive transfusion in Cambodia), serology (S. mekongi) + but also hepatitis B or C +, hepatic biological exams (hepatic insufficiency). So we decided for eleven of them to use a surgical decompression procedure in order to decrease portal hypertension and the porto-systemic gradient. After defining portal hypertension, specific clinical features of portal hypertension (secondary to Schistosomiasis) the authors report eleven cases who were operated on (2000-2002): 4 mesenterico-cave shunt with interposition of a graft (Drapanas' procedure), 1 operation of HASSAB (after splenectomy), 6 proximal spleno-renal diversion (after splenectomy). After studying the results of the eleven patients, discussion with other surgical procedures, particularly endoscopic procedures is developed. The follow-up of these patients during at least five years is mandatory to give guidelines for post-systemic shunts to prevent rebleeding (near other methods). Treated too late, schistosomiasis has no benefit from drugs (Praziquentel). After a mean period of forty two months, the following results are: mortality: one case (10 days after operation): hepatic insufficiency (group Child B/C). morbidity: one occlusion of the small intestine, after 4 months (debridment), operated at the Provincial hospital of Kratie (case no1). Ten patients resume work, family and social life between Kratie and Sambor in 2002. No rebleeding. No encephalopathy.

Investigation of bioavailability, pharmacokinetics and safety of new pediatric formulations of artesunate and mefloquine.

The bioavailability/pharmacokinetics of dihydroartemisinin and mefloquine following the oral doses of 4 mg/kg body weight artesunate (Cambodian Pharmaceutical Enterprise) given concurrently with 10 mg/kg body weight oral mefloquine artesunate (Cambodian Pharmaceutical Enterprise) were investigated in 15 healthy Cambodian male volunteers. Both formulations were generally well tolerated. Both produced satisfactory plasma/blood concentration-time profiles. Oral artesunate and mefloquine were rapidly absorbed from gastrointestinal tract with marked inter-individual variation. For the dihydroartemisinin, the median (95% Cl) Cmax of 748 (304-1,470) ng/ml was observed at 1.5 (0.3-3.0) hours (tmax) after drug administration. The median (95% CI) values for AUC0-infinity, lambda(z) and tl/2z were 1.673 (1.08-2.88) microg.h/ml, 0.54(0.24-1.1)/hour and 1.3 (0.6-2.9) hours, respectively. For mefloquine, a median (95% Cl) Cmax of 1,000 (591-1,500) ng/ml was observed at 4 (2-6) hours (tmax) after drug administration. The median (95% CI) value for AUC0-168h was 3.92 (2.88-7.02) microg.h/ml.

Presence of Anopheles culicifacies B in Cambodia established by the PCR-RFLP assay developed for the identification of Anopheles minimus species A and C and four related species.

A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay developed for identification of five species of the Anopheles minimus Theobald group and a related mosquito species of the Myzomyia Series (Diptera: Culicidae) was applied to morphologically identified adult female specimens collected in Ratanakiri Province, north-eastern Cambodia. In addition to finding An. aconitus Dönitz, An. minimus species A and An. pampanai Büttiker & Beales, some specimens showed a new restriction banding pattern. Siblings of specimens that exhibited this new PCR-RFLP pattern were morphologically identified as An. culicifacies James sensu lato. Based on nucleotide sequences of the ribonuclear DNA internal transcribed spacer 2 region (ITS2) and the mitochondrial cytochrome oxidase I gene (COI), these specimens were recognized as An. culicifacies species B (sensu Green & Miles, 1980), the first confirmed record of the An. culicifacies complex from Cambodia. This study shows that the PCR-RFLP assay can detect species not included in the initial set-up and is capable of identifying at least seven species of the Myzomyia Series, allowing better definition of those malaria vector and non-vector anophelines in South-east Asia.

Epidemiology and control of mekongi schistosomiasis.

Since the first case of Schistosoma mekongi infection was reported in 1957, control measures have been implemented in Laos and in Cambodia. Operational research provided the necessary information on parasite epidemiology and the associated morbidity in order to develop adequate control measures. S. mekongi transmission occurs in rocky banks of the river according to a seasonal cycle. Common daily activities of villagers living in the endemic areas constitute the risk factors for infection. The potential role of an animal reservoir is not fully understood. Severe disease is associated with advanced infection status. Signs and symptoms of portal hypertension dominate the clinical situation, and death is usually due to bleeding from ruptured esophageal varices. Schistosomiasis control in both Laos and Cambodia was based on universal treatment campaigns and resulted in a dramatic fall in the prevalence of the infection and in morbidity control. However, even if the disease and the infection have been satisfactorily controlled, transmission still occurs, and in very limited areas the prevalence reaches rates of more than 15%. Today, 60000 people are estimated to be still at risk of infection in Laos and about 80,000 in Cambodia. The new challenge in schistosomiasis control in endemic areas along the Mekong river is to consolidate the results, to establish a sensitive and reliable surveillance system, and finally to adapt control strategies to the low endemic situation. The impoverished nature of the region makes the possibility of sanitation unfeasible, and external support is still needed to sustain activities in the near future and to enable the substantial reduction of risk behaviors.