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

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 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 countrie

Socially-marketed rapid diagnostic tests and ACT in the private sector: ten years of experience in Cambodia.

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

National malaria prevalence in Cambodia: microscopy versus polymerase chain reaction estimates

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

Molecular Surveillance for Multidrug-Resistant Plasmodium falciparum, Cambodia

We conducted surveillance for multidrug-resistant Plasmodium falciparum in Cambodia during 2004–2006 by assessing molecular changes in pfmdr1. The high prevalence of isolates with multiple pfmdr1 copies found in western Cambodia near the Thai border, where artesunate–mefloquine therapy failures occur, contrasts with isolates from eastern Cambodia, where this combination therapy remains highly effective

Intrahost modeling of artemisinin resistance in Plasmodium falciparum

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 log10 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

Production and validation of durable, high quality standardized malaria microscopy slides for teaching, testing and quality assurance during an era of declining diagnostic proficiency

Background: Sets of Giemsa-stained, blood smear slides with systematically verified composite diagnoses would contribute substantially to development of externally validated quality assurance systems for the microscopic diagnosis of malaria. Methods: whole blood from Plasmodium-positive donors in Cambodia and Indonesia and individuals with no history of risk for malaria was collected. Using standard operating procedures, technicians prepared Giemsastained thick and thin smears from each donor. One slide from each of the first 35 donations was distributed to each of 28 individuals acknowledged by reputation as having expertise in the microscopic diagnosis of malaria. These reference readers recorded presence or absence of Plasmodium species and parasite density. A composite diagnosis for each donation was determined based on microscopic findings and species-specific small subunit ribosomal RNA (ssrRNA) DNA polymerase chain reaction (PCR) amplification. Results: More than 12, 000 slides were generated from 124 donations. Reference readers correctly identified presence of parasites on 85% of slides with densities \u3c100 parasites/μl, which improved to 100% for densities \u3e350 parasites/μl. Percentages of agreement with composite diagnoses were highest for Plasmodium falciparum (99%), followed by Plasmodium vivax (86%). Conclusion: Herein, a standardized method for producing large numbers of consistently high quality, durable Giemsa-stained blood smears and validating composite diagnoses for the purpose of creating a malaria slide repository in support of initiatives to improve training and competency assessment amidst a background of variability in diagnosis is described

Production and validation of durable, high quality standardized malaria microscopy slides for teaching, testing and quality assurance during an era of declining diagnostic proficiency

Background: Sets of Giemsa-stained, blood smear slides with systematically verified composite diagnoses would contribute substantially to development of externally validated quality assurance systems for the microscopic diagnosis of malaria. Methods: whole blood from Plasmodium-positive donors in Cambodia and Indonesia and individuals with no history of risk for malaria was collected. Using standard operating procedures, technicians prepared Giemsastained thick and thin smears from each donor. One slide from each of the first 35 donations was distributed to each of 28 individuals acknowledged by reputation as having expertise in the microscopic diagnosis of malaria. These reference readers recorded presence or absence of Plasmodium species and parasite density. A composite diagnosis for each donation was determined based on microscopic findings and species-specific small subunit ribosomal RNA (ssrRNA) DNA polymerase chain reaction (PCR) amplification. Results: More than 12, 000 slides were generated from 124 donations. Reference readers correctly identified presence of parasites on 85% of slides with densities \u3c100 parasites/μl, which improved to 100% for densities \u3e350 parasites/μl. Percentages of agreement with composite diagnoses were highest for Plasmodium falciparum (99%), followed by Plasmodium vivax (86%). Conclusion: Herein, a standardized method for producing large numbers of consistently high quality, durable Giemsa-stained blood smears and validating composite diagnoses for the purpose of creating a malaria slide repository in support of initiatives to improve training and competency assessment amidst a background of variability in diagnosis is described

Single-nucleotide polymorphism, linkage disequilibrium and geographic structure in the malaria parasite Plasmodium vivax: prospects for genome-wide association studies

<p>Abstract</p> <p>Background</p> <p>The ideal malaria parasite populations for initial mapping of genomic regions contributing to phenotypes such as drug resistance and virulence, through genome-wide association studies, are those with high genetic diversity, allowing for numerous informative markers, and rare meiotic recombination, allowing for strong linkage disequilibrium (LD) between markers and phenotype-determining loci. However, levels of genetic diversity and LD in field populations of the major human malaria parasite <it>P. vivax </it>remain little characterized.</p> <p>Results</p> <p>We examined single-nucleotide polymorphisms (SNPs) and LD patterns across a 100-kb chromosome segment of <it>P. vivax </it>in 238 field isolates from areas of low to moderate malaria endemicity in South America and Asia, where LD tends to be more extensive than in holoendemic populations, and in two monkey-adapted strains (Salvador-I, from El Salvador, and Belem, from Brazil). We found varying levels of SNP diversity and LD across populations, with the highest diversity and strongest LD in the area of lowest malaria transmission. We found several clusters of contiguous markers with rare meiotic recombination and characterized a relatively conserved haplotype structure among populations, suggesting the existence of recombination hotspots in the genome region analyzed. Both silent and nonsynonymous SNPs revealed substantial between-population differentiation, which accounted for ~40% of the overall genetic diversity observed. Although parasites clustered according to their continental origin, we found evidence for substructure within the Brazilian population of <it>P. vivax</it>. We also explored between-population differentiation patterns revealed by loci putatively affected by natural selection and found marked geographic variation in frequencies of nucleotide substitutions at the <it>pvmdr-1 </it>locus, putatively associated with drug resistance.</p> <p>Conclusion</p> <p>These findings support the feasibility of genome-wide association studies in carefully selected populations of <it>P. vivax</it>, using relatively low densities of markers, but underscore the risk of false positives caused by population structure at both local and regional levels.</p> <p>See commentary: <url>http://www.biomedcentral.com/1741-7007/8/90</url></p