Methods Study Area This study was conducted in the Gebiley District in Somaliland in northwestern Somalia. The district has a pre- dominantly arid landscape with a few seasonal rivers and patches of irrigated farmlands. It is an area of intense sea- sonal rainfall with an average annual precipitation of 59.9 mm (2004–2007) and 2 peaks in rainfall in April and Au- gust. Three moderately sized communities were randomly selected from census maps by using spatial random sampling techniques in Arcview version 3.2 (Environmental Systems Research Institute, Redlands, CA, USA) (16). These com- munities were the villages of Xuunshaley (9.72140°N, 43.42416°E), Badahabo (9.68497°N, 43.65616°E), and Ceel-Bardaale (9.81777°N, 43.47455°E). The research protocol was reviewed and approved by the Research Eth- ics Review Committee of the World Health Organization (RPC246-EMRO) and the Ethical Committee of the Minis- try of Health and Labor, Republic of Somaliland. Data Collection Two cross-sectional surveys were conducted. The ﬁrst survey was conducted in March 2008 to determine parasite carriage at the end of the dry season (16). The purpose of the survey and the procedures were ﬁrst discussed with the clan elders; thereafter, each household was visited, and in- formed consent was sought from each head of household. Households that agreed to participate were geolocated by using a global positioning system (Garmin eTrex; Garmin International, Inc., Olathe, KS, USA), and information was collected on demographic characteristics, bed net use, and travel history of the participants. Distance to seasonal rivers or other water bodies and distance to the nearest livestock enclosure was determined by using the global positioning system. Individual written consent was obtained from all liter- ate adults; illiterate adults provided consent by a thumbprint in the presence of an independent literate adult witness. For children <18 years of age, consent was obtained from parents or guardians, and children 12–18 years of age who could not write also provided consent by a thumb print. One ﬁngerprick blood sample was obtained from each respondent for the preparation of a P. falciparum antigen– speciﬁc rapid diagnostic test (RDT) (Paracheck-Pf; Orchid Biomedical Systems, Goa, India) sample and thick and thin blood smears. One-hundred high-power microscopic ﬁelds were examined and an additional 100 ﬁelds were exam- ined if the ﬁrst 100 ﬁelds were negative. RDT results were used for treatment with sulfadoxine-pyrimethamine and 3 doses of artesunate according to national guidelines. A sec- ond cross-sectional survey was conducted at the end of the wet season (August–September 2008) by using procedures Serologic Markers for Detecting Malaria identical to those described above, except that part of the ﬁngerprick blood sample was placed on ﬁlter paper (3 MM; Whatman, Maidstone, UK) as described by Corran et al. (17). Entomologic Surveys Presence of Anopheles spp. mosquitoes in the area was determined by larvae collections in all permanent water bodies (artiﬁcial rain water reservoirs, wells, boreholes, stagnant storage pits, and riverbeds) in the 3 villages at the end of the wet season. Locally produced 250-mL dippers with a white surface were used. Five to 10 dips were made in the large water bodies and the presence of Anopheles spp. larvae was visually assessed and recorded. Elution of Serum Filter paper samples were stored at 4°C with desiccant until processed. A 3.5-mm blood spot, equivalent to ≈3 μL of blood (17), was punched from the ﬁlter paper and placed in a labeled well of a low-binding 96-well titer plate. A to- tal of 300 μL of reconstitution buffer (phosphate-buffered saline [PBS], 0.05%Tween, and 0.1% [wt/vol] sodium az- ide) was added, and plates were sealed and rocked gently at room temperature overnight and subsequently stored at 4°C. The reconstituted blood spot solution was equivalent to a 1:100 dilution of whole blood or a 1:200 dilution of serum. ELISAs All reconstituted ﬁlter paper spots were tested at a ﬁnal serum dilution equivalent of 1:1,000 for human im- munoglobulin G antibodies against P. falciparum mero- zoite surface protein 119 (MSP-119) and 1:2,000 for anti- bodies against apical membrane antigen 1 (AMA-1) by using described ELISA methods (12,17). Brieﬂy, recom- binant MSP-119 (Wellcome genotype) and AMA-1 (3D7) were coated overnight at 4°C at a concentration of 0.5 μg/ mL. Plates were washed by using PBS, 0.05% Tween 20 (PBS/T) and blocked for 3 h with 1% (wt/vol) skim milk powder in PBS/T. Positive controls (a pool of hyperim- mune serum) and negative controls (European malaria- negative volunteers) were added in duplicate to each plate. The plates were washed and horseradish peroxidase–conju- gated rabbit anti-human immunoglobulin G (Dako, Rosk- ilde, Denmark) (1:5,000 dilution in PBS/T) was added to all wells. Plates were developed for 20 min by using an o-phenylenediamine dihydrochloride substrate solution. Reactions were stopped with 2 mol/L H2SO4. Plates were read immediately at 492 nm and optical density (OD) val- ues recorded. For P. vivax, an identical protocol was used with MSP-119 (0. 5 μg/mL) (18) and AMA-1 (0. 5 μg/mL). Serum in this protocol was used at 1:1,000 dilutions for both antigens. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 16, No. 3, March 2010 393