FEBRILE SURVEILLANCE IN CAMBODIA 247 were added: two in August 2007, one in October 2007, one in December 2007, one in February 2007, one in March 2008, and one in April 2008, for a total of nine. Five of the health care sites were located in Operational District A (peri-urban); four health care sites were located in Operational District B (rural). All health care sites were located within 50 km of Phnom Penh in south-central Cambodia (Figure 2). Patients were asked to enroll if they meet the inclusion cri- teria, which included: a measured tympanic membrane tem- perature > 38.0 C when at the study site and, as reported by the enrollee, a fever duration of at least 24 hours and < 10 days, were 2 years of age or older, and after medical examination, their illness was not able to be diagnosed during the patient’s physical examination or medical history.5 Patients were excluded if they declined to participate in the study or did not meet any of the inclusion criteria. A physician or medical assistant in each clinic obtained written informed consent, administered a pretested enrollment questionnaire, and performed a medical examination. Laboratory technicians collected blood and throat and nasal swabs. All study participants were directed to return 14 to 21 days for a follow-up appointment, in which a convalescent blood specimen was collected (Figure 1). All clinical specimens were transported to a central laboratory where testing for several infectious agents was performed. Definitions. Influenza-like illness was defined by fever (> 38.0 C), plus cough or sore throat.11–13 Diarrhea was defined as two or more loose liquid stools in any 24-hour period as all enrolled patients had the symptom of fever. Probable dengue was defined as only having a positive immunoglobulin M (IgM) result and confirmed dengue was defined as having a positive polymerase chain reaction (PCR) result. Recent travel was defined as the individual leaving their respective home for at least 5 consecutive days within 2 months preceding enrollment. Operational district is a designation used by the Cambodian FIGURE 1. Flow chart of patient enrollment, information collected, and testing algorithms. government to denote a continuous geographic region under the purview of a single government office. Specimen collection. For each enrollee, a blood specimen was obtained by venipuncture and one throat and one nasal swab were collected. For nasal swabs, a dry polyester swab was inserted into the nostril, parallel to the palate, slowly withdrawn, and placed in a vial containing 2–3 mL of virus transport medium. For throat swabs, both tonsils and the posterior pharynx were swabbed vigorously, and the swab placed in 2–3 mL of virus transport medium. All inoculated vials were kept at 4 C until transported to NAMRU2/NIPH’s laboratory. All specimens were accessioned at the laboratory between 24 and 72 hours post collection. Malaria smears. At the time of enrollment a blood smear is prepared and transported (daily) to the laboratory for staining by Giemsa stain and interpretation by microscopy. Blood cultures. A single blood culture was obtained from all enrolled patients in an aerobic BacT/Alert 3D (bioMerieux, Hazelwood, MO) bottle. Daily, inoculated bottles were transported to the NAMRU2 laboratory in Phnom Penh and then incubated for up to 7 days in the BacT/Alert blood culture system. A sample of blood from positive blood cultures was inoculated onto chocolate, MacConkey’s, and blood agar plates and incubated at 37 C, 5% CO2, for up to 48 hours. Bacterial identification was done using the API 20E biochemical identification system (bioMerieux). Serological assays. All commercial enzyme-linked immu- nosorbent assay (ELISA) test assays were performed and interpreted according to the manufacturer’s instruction as Positive, Equivocal, and Negative. Dengue IgM serological assays were performed on all acute serum samples from all study participants using the Dengue IgM Capture ELISA Dx Select (Focus Diagnostics, Cypress, CA). Hepatitis A IgM was performed with the Hepalisa Anti-HAV IgM (PT INDEC DIAGNOSTICS, Jakarta, Indonesia). Hepatitis B surface antigen was detected using the Hepalisa HBsAg (PT INDEC DIAGNOSTICS). An IgM serological response to hepatitis E was detected using the HEV IgM ELISA 3.0 (MP Biomedicals, Singapore). An acute immunological response was detected to Leptospira using the Leptospira IgM ELISA (PANBIO, Brisbane, Australia). The IgG serology was performed for dengue, Hantavirus, chikungunya, and Rickettsia on all convalescent serum sam- ples. All commercial ELISA test assays were performed and interpreted according to the manufacturer’s instruction as Positive, Equivocal, and Negative. Convalescent specimens that were found to be IgG positive were paired with the acute serum and retested. A 4-fold increase in titer from acute to convalescent sample, or a result change going from nega- tive to positive, was considered indicative of seroconversions. Detection of dengue IgG and hantavirus IgG were accom- plished by using the Dengue IgG capture ELISA (PANBIO) and Hantavirus IgG ELISA Dx Select (Focus Diagnostics), respectively. Chikungunya virus-specific IgG was detected by ELISA as previously published.14 The IgG ELISA specific for typhus group and scrub typhus rickettsiae and spotted fever group ricketssiae were performed as previously described.15,16 A 4-fold increase in titer from acute to convalescent sample, or a result change going from negative to positive, was consid- ered indicative of seroconversions. PCR amplification. Influenza. The RNA was extracted from nasal and throat swabs using QIAamp viral RNA mini