FEBRILE SURVEILLANCE IN CAMBODIA 249 of DNA, 12.5 mL of 2 +TaqMan Universal PCR Master mix (Cat. no. ABI 4304437), 0.4 mM for each primer and 0.3 mM of probe; and the R. felis PCR assay contained 2 mL of DNA, 12.5 mL of 2 +TaqMan Universal PCR Master mix (Cat. no. ABI 4304437), 0.4 mM for each primer and 0.4 mM of probe. The Applied Biosystems 7500 real time thermocycler (Applied Biosystems) was used for all real-time PCR assays. The thermocycling program used consisted of the following parameters; 50 C for 2 minutes, 95 C for 10 minutes, and 60 cycles with 95 C for 15 seconds, 60 C for 30 seconds. rRT-PCR for hantaviruses. Hantavirus RNA was extracted from serum using QIAamp viral RNA mini kits (QIAGEN) following manufacturer’s instruction and stored at 80 C. An rRT-PCR assay was used to detect Hantaviruses serotypes Hantaan (HTN), Puumala (PUU), Seoul (SEO), and Dobrava- Belgrade (DOB).21 One-step rRT-PCR was performed in a final volume of 25 mL containing 5 mL of extracted RNA, 12.5 mL of buffer mix, 0.5 mL of MgSO4, and 0.625 mL Superscript III/Platinum Taq-Enzyme mix, 0.5 mL of RNAse- out (Invitrogen), 0.4 mM for each primer, and 0.25 mM of probe. The Rotor-Gene 6000 real time thermocyler (Corbett Life Science, Sydney, Australia) was used for all PCR reactions. The thermocycling parameters for all targets consisted of 50 C for 15 minutes, 95 C for 2 minutes, and 45 cycles with 95 C for 15 seconds, 55 C for 30 seconds, and 72 C for 1 minute. Nested RT-PCR for detection of hepatitis E viruses. The RNA was extracted from serum using QIAamp viral RNA mini kits (QIAGEN) following manufacturer’s instruction and stored at 80 C. The nonstructural (NS) gene of Hepatitis E virus was detected using a nested reverse real-time PCR assay.22 The RT reactions were performed in 25 mL containing 5 mL of Qiagen buffer mix (Qiagen, Cat. no. 210212) and 1 mL of enzyme mix, 1 mL of 10 mM dNTP, 0.63 mM of each primer, 0.15 mL of 40 U RNase inhibitor (Promega, Cat. no. N2511), and 5 mL of RNA template. The PCR thermal cycling was performed as follows: 45 C for 30 minutes, 94 C for 15 minutes, and 40 cycles with 94 C for 1 minute, 45 C for 1 minute, 72 C for 1 minute, and then 72 C for 7 minutes. The PCR reactions were performed using the Applied Biosystems 9700 (Applied Biosystems). An nPCR reaction was carried out at a total volume of 25 mL. The reaction contained 2.5 mL of ABI PCR buffer (Applied Biosystems, Cat. no. N808-0009), 1.5 mM MgCl2, 2 mL of 10 mM dNTP, 0.75 mM forward primer and reverse primer, 0.125 mL of 5 U of AmpliTaq DNA Polymerase, and 5 mL of diluted (1:50) RT-PCR product. The cycle conditions of nPCR reaction are as follows: 94 C for 2 minutes, and 40 cycles with 94 C for 1 minute, 55 C for 1 minute, 72 C for 1 minute, and then 72 C for 7 minutes. Products were visualized on 2% agarose gels stained with ethidium bromide under UV light. Statistical analysis. All data was double-data entered into MS Access (Microsoft Inc., Redmond, WA). Data was imported into SAS v 9.1 (SAS, Cary, NC), which was used for all statistical analyses. RESULTS From December 2006 through December 2009, 9,997 patients were enrolled (Figure 3). All patients enrolled met the criteria of fever (> 38.0 C). Among the 9,997 patients, 89.3% had a complete questionnaire. A serum sample, nose and/or throat swab, and blood culture sample was available from 99.8%, 99.7%, and 98.2%, respectively. The median age of enrolled patients was 13 years of age (interquartile range [IQR] 6–28) years of age (Table 1) and 53.9% were male. Patients reported both recent travel to other provinces or countries (7.9%) and travel into forested areas (7.1%). Study subject occupation was recorded, however stu- dents and children accounted for 63.5% of enrolled study sub- jects. Among study subjects reporting a specific occupation, grain farmers were most common (31.3%). Contact with cat- tle (8.3%), chickens (7.7%), and dogs (7.0%) were the most common reported animal exposures. Prior medication usage was reported by 36.6% of enrolled subjects. The most common symptoms reported by enrolled patients included headache (69.5%), cough (57.5%), sore throat (46.6%), chills (48.1%), and malaise (46.8%) (Table 1). Among enrolled participants, 65.7% presented with signs and symptoms characteristic of influenza-like illness, 17.1% with gastroenteritis, and 1.4% with jaundice. Close contact with an individual who had sim- ilar symptoms was reported among 9.0% of participants. Of the enrolled patients, 10.1% were hospitalized or referred for hospitalization. Laboratory testing was performed for agents believed to be endemic to the region in addition to a variety of emerging pathogens (Table 2). Of enrolled patients, 19.9% (N ¼ 1,983) were PCR positive for influenza. Dengue virus was considered the probable cause of illness (as defined by only a positive IgM result) in 842 enrollees (8.4%) and confirmed (as defined by a positive PCR result) in 883 cases (8.9%). All four serotypes of dengue were detected during the study period. Malaria was identified among 7.2% (N ¼ 716) of patients, with Plasmodium falciparum identified in 30.2% of malaria cases. In addition, a total of 19 mixed infections of Plasmodiuom vivax and P. fal- ciparum were identified. Acute leptospirosis was serologically identified in 20.8% of study subjects. Hepatitis A IgM testing was performed on the first 3,243 patients enrolled, 56 (1.7%) of the patients were positive. Hepatitis B surface antigen test- ing was done on the first 4,596 enrollees, and 261 (5.8%) were positive. Follow-up on testing of HBsAG-positive samples identified eight samples that were also positive for IgM. A hepatitis E IgM response was detected among 11.1% of par- ticipants. The PCR testing of 517 serum samples that were pos- itive or equivocal for Hepatitis E IgM identified five samples positive for hepatitis E nucleic acid. Serological testing of paired samples was conducted for anti- bodies against chikungunya, hantavirus, and rickettsiae (scrub typhus group, typhus group, and spotted fever group) (Table 3). Testing of chikungunya IgG among the first 753 patients enrolled in 2007 identified a previous exposure in 16.3%. Chikungunya IgM was identified among 0.1% (2 of 1,438) of enrolled patients and PCR of patients presenting with fever and joint pain identified three cases out of 571 samples tested. Hantavirus IgG was detected in convalescent specimens of 8.1% of patients tested. Paired testing of acute and convales- cent samples identified 71 patients that had evidence of an acute hantavirus infection. No IgM-positive or PCR-positive cases of hantaviral infection were identified by IgM or molec- ular analysis. Testing for scrub typhus group, typhus group, and spotted fever group rickettsiae-specific IgG in convalescent specimens identified seroprevalences of 7.0%, 13.4%, and 11.6%, respec- tively (Table 3). From positive convalescent samples, testing of paired samples to identify acute cases identified 35, 56, and 21 cases of scrub typhus, typhus, and spotted fever, respectively.