Lumefantrine, Artemether

Satellite images were used to construct a sampling frame for the random selection of households enrolled in prospective longitudinal and cross-sectional surveys of malaria parasitaemia in the catchment area of Macha Hospital in Southern Province, Zambia (Figure 1). Macha Hospital is approximately 70 km from the nearest town of Choma and the catchment area is populated by traditional villagers living in small, scattered homesteads. Anopheles arabiensis is the primary vector responsible for malaria transmission, which peaks during the rainy season from December through April [11 (link)]. The sampling frame for the random selection of households was constructed from a Quickbird™ satellite image obtained from DigitalGlobe Services, Inc. (Denver, Colorado). The image was imported into ArcGIS 9.2 (Environmental Systems Research Institute [ESRI], Redlands, California) and locations of households were identified and enumerated manually. Structures of appropriate size and shape were identified as potential residences, and consisted of one or more domestic structures where members of a family resided. Smaller structures, such as kraals, and larger structures, such as schools, were excluded. Selected households were allocated to one of two study cohorts: longitudinal and cross-sectional. Households in the longitudinal cohort were surveyed repeatedly approximately every two months and households in the cross-sectional cohort were surveyed once. Cross-sectional and longitudinal household surveys were conducted approximately every other month (during alternate months) from April 2007 through December 2007 in the first study area and from February 2008 through December 2008 in the second study area. Data from all cross-sectional households and the first longitudinal household visit were used to develop the spatial risk model. Model validation was conducted with the full longitudinal dataset. The study was approved the Johns Hopkins Bloomberg School of Public Health Institutional Review Board and the University of Zambia Research Ethics Committee.
A field team was provided with images and coordinates of the randomly selected households. After obtaining permission from the local chief and head of household, and individual written informed consent, a questionnaire was administered to each participant residing within the household and a blood sample was collected by finger prick. Rapid diagnostic tests (RDT; ICT Diagnostics, Cape Town, South Africa) were used to detect P. falciparum histidine-rich protein 2. This RDT was shown to detect 82% of test samples with wild-type P. falciparum at a concentration of 200 parasites/μL 98% of test samples with a concentration of 2,000 parasites/μL, with false positives in 0.6% of clean negative samples [12 ]. Individuals who were RDT positive were offered treatment with artemether-lumefantrine (Coartem^®) by trained medical personnel. Households in which at least one individual tested positive by RDT were classified as a positive household. Positive and negative households were plotted as a data layer in ArcGIS 9.2.

The cohorts under surveillance for malaria episodes were located in Chonyi, Ngerenya, and Junju sublocations of Kilifi District, on the coast of Kenya between January 1998 and June 2009 (Figure 1). Concurrent entomological studies and parasite prevalence rates suggest that the transmission intensity is higher in Junju and Chonyi than in Ngerenya [33] (link),[34] (link), but transmission has been falling throughout the period of study [35] (link).
The field methods used to identify episodes of febrile malaria and asymptomatic parasitaemia have previously been described [36] (link),[37] (link). Weekly active surveillance was undertaken, and children with fever had blood slides for malaria parasites. In Chonyi and Ngerenya, children with either subjective (i.e., reported) or objective fever (temperature ≥37.5°C) had blood smears performed for estimating the parasite density. In Junju blood smears were done only on children with an objective fever, but children with subjective fever were seen again 6–12 h later, and the temperature measurement repeated. Blood smears were made if objective fever was confirmed at this measurement.
The parents of the children in Chonyi and Ngerenya were instructed to report to Kilifi District Hospital 20 km away if the child had any symptoms of disease at any time (and reimbursed for travel expenses), and in Junju trained field workers were available at all times in the villages for passive surveillance. Antimalarials were supplied for proven episodes of malaria by the study team in accordance with government of Kenya guidelines; this was sulfadoxine-pyrimethamine until 2004, and co-artemether thereafter. Study participants may have used private clinics or bought antimalarials without the study team's knowledge, but given the availability of free treatment this was probably infrequent.
Surveys for asymptomatic parasitaemia were undertaken once yearly, immediately before the rainy season. All individuals recruited to the study were asked to attend for blood sampling, and microscopy results were available for 70%–88% of participants for each survey.
The Geographic Positioning System coordinates from the Kilifi Demographic Surveillance Survey were linked to each homestead in the study. In Ngerenya and Chonyi, all the residents at individual homesteads were recruited, but in Junju only children under 8 y of age were recruited. The homesteads in Ngerenya and Junju were evenly spaced throughout the study location, but in Chonyi the homesteads were distributed along a central road through the study area. Children born in the study homesteads during the period of monitoring were recruited, and so the average age of the cohort did not increase over time (Table 1).

This was a longitudinal, single-arm, prospective study to evaluate P. falciparum tolerance to ART and its derivatives in children with uncomplicated malaria aged 6 months to 14 years in 3 health facilities in the Greater Accra region of Ghana. The study focused mainly on day 3 post artemether-lumefantrine (AL) treatment parasitaemia, 72-h ex vivo RSA after dihydroartemisinin (DHA) exposure, 72-h parasite clearance in vitro against a panel of 6 drugs (ART, AS, artemether [AM], DHA, amodiaquine [AQ], lumefantrine [LUM], and the following molecular markers of drug tolerance / resistance: Single Nucleotide Polymorphisms (SNPs), Multiple Nucleotide Polymorphisms (MNPs), Insertions & Deletions (INDEL) in Pfk13, Pfcoronin, P. falciparum multidrug resistance protein 1 (Pfmdr1), multidrug resistance protein 2 (Pfmdr2), dihydrofolate reductase (Pfdhfr), dihydropteroate synthetase (Pfdhps), signal peptide peptidase (Pfspp), and multidrug resistance-associated protein 2 (Pfmrp2) genes. It sought to set up correlates of ART tolerance.

Individuals who visited the Service de Lutte Antipaludique (SLAP) clinic with signs and symptoms suggestive of uncomplicated P. falciparum malaria were screened by microscopic examination on Giemsa-stained blood smears. Malaria infected patients were treated with artemether-lumefantrine (AL, Coartem), according to the treatment guideline of the Senegal National Malaria Control Program (NMCP). For each patient, 5 mL vacutainer tubes of venous blood were collected for ex vivo RSA.
Parasite culture and ring-stage survival assay. Parasitemia was estimated by microscopy examination on Giemsa-stained blood smears. Venous blood samples were then processed by eliminating plasma, leukocytes and anticoagulant from red blood cells (RBCs), washed twice in RPMI 1640 medium (Gibco, Life technologies). Parasitemia were adjusted to 1% if greater by adding uninfected RBCs as previously described [10 (link)]. Then, 900 μL RBCs were loaded into wells, exposed to either 100 μL of 700 nM DHA or0.1% of dimethyl sulfoxide (DMSO) and cultivated at 37°C in incubator for six hours (conditions: 94% N₂, 5% CO₂, 1% O₂) [30 (link)]. Finally, RBCs were washed and cultivated for 66 hours. The proportions of viable parasites were estimated independently by two expert malaria microscopists on Giemsa-stained thin smears. The number of viable parasites that developed into ring/trophozoite stages were determined, pyknotic forms were excluded. The average of the two counts was calculated. If any discrepancy was noted (either difference of parasite density of > 50%), slides were checked by a third independent reader, and parasite densities were calculated by averaging the two most close counts. Survival rates were calculated as the ratio of parasites in exposed and non-exposed cultures. Results were deemed as interpretable if the parasitemia in the sample exposed to DMSO was higher than the initial parasitemia at 0h [31 ].