FEBRILE SURVEILLANCE IN CAMBODIA 251 TABLE 2 Distribution of pathogens identified among enrolled patients from Dec. 2006 to Dec. 2009 Pathogens Tested cases Positive (N) Positive (%) Influenza viruses H1N1 H3N2 H5N1 Pandemic H1N1 Influenza B Influenza A, untyped H1N1 and influenza B H3N2 and influenza B Pandemic H1N1 and H3N2 Malaria P. falciparum P. vivax P. falciparum and P. vivax Dengue IgM positive only (probable dengue) PCR positive (confirmed dengue) Serotype-1 Serotype-2 Serotype-3 Serotype-4 Blood-borne pathogen Leptospirosis IgM Hepatitis A IgM Hepatitis B sAG Hepatitis B IgM Hepatitis E IgM Hepatitis E PCR Chikungunya IgG (convalescent) Chikungunya IgM Chikungunya PCR Hantavirus IgG (convalescent) Hantavirus 4 +IgG rise and/or seroconverter Hantavirus PCR Scrub typhus group IgG (convalescent) Scrub typhus group-specific IgG 4-fold rise in titer or seroconversion Orientia tsutsugamushi-specific qPCR Typhus group IgG (convalescent) Typhus group-specific IgG 4-fold rise in titer or seroconversion R. typhi-specific qPCR Spotted fever group IgG (convalescent) Spotted fever group-specific IgG 4-fold rise in titer or seroconversion Tick-borne rickettsiae-specific qPCR presenting for treatment at clinics in south-central Cambodia. This is the first comprehensive study to identify etiologies of AFI in the general population of Cambodia. Our study enrolled nearly 10,000 patients over a 3-year period. During this time period, there was a seasonality to enrollment that corresponded to the rainy season, which TABLE 3 Pathogens identified from testing of acute and convalescent serum samples Pathogens Tested cases Positive (N) Positive (%) 1,983 224 827 1 286 627 18 1 3 4 716 216 481 19 842 883 83 398 108 294 629 2,076 58 267 8 1038 5 123 2 3 459 71 0 133 35 2 261 56 1 146 21 0 19.9 11.3 41.7 < 0.1 14.4 31.6 0.9 < 0.1 0.2 0.2 7.2 30.2 67.2 2.6 9.4 45.1 12.2 33.3 6.3 20.8 1.7 5.8 3.0 11.1 1.0 16.3 0. 1 0.5 8.1 15.5 0.0 7.0 26.3 3.0 13.4 21.5 1.2 11.6 14.4 0.0 9,968 9,954 9,975 9,821 9,975 3,243 4,596 267 9,353 517 753 1438 571 5675 459 79 1906 133 67 1946 261 83 1263 146 37 roughly corresponds with the influenza season in Cambodia23 peaking in August–October of each year. In this study, we identified influenza, Leptospira, dengue, hepatitis E, and malaria as the most frequently identified pathogens associated with AFI among Cambodians. Previous regional studies had identified similar etiologies among patients with AFI,5,6 with some variation of etiologies and dis- tribution that could be based on the sampled population or the pathogen testing schemes. However, we identified that many of the acute serological responses to Leptospira and hepatitis E were associated with co-detection of other organisms (data not shown). A large number of study subjects were classified chronic hepatitis B cases; additional testing revealed that only a small number of those individuals presented with an AFI caused by hepatitis B. A recent study of hepatitis B among blood donors found a similar percentage of HBsAG-positive volunteers24 in Cambodia. Despite a lack of jaundice reported among the study popu- lation, a high percentage of patients showed an acute serologi- cal response to hepatitis E. Of the 175 patients presenting with clinical jaundice, only 29 (16.6%) were identified to have a positive hepatitis E IgM response. This represents a diagnostic challenge that may be caused by a long-term IgM response or continuous exposure in the environment to hepatitis E virus. A large number of patients were positive for an acute sero- logical response to Leptospira. A recent study focusing on hos- pitalized patients in Cambodia found a similar prevalence of acute or prior Leptospira infection, with approximately half of the patients presenting with an acute infection.25 Although lep- tospirosis has been documented to be endemic in the region,26 the more definitive microscopic agglutination testing (MAT) was not performed on these samples. In addition, a recent study showed poor diagnostic accuracy of the Leptospira IgM PanBio ELISA compared with MAT testing.27 Further labora- tory studies and analysis is needed to understand the contribu- tion of Leptospira to AFI in Cambodia. Almost 1% of enrollees tested had results suggesting an acute hantavirus infection. However, PCR testing failed to identify Hantaan, Seoul, Puumala, or Dobrava strains among any study subjects. This may reflect genetic differ- ences of Hantavirus strains in Cambodia that need to be fur- ther defined. Alternatively, the serology results may be from a cross-reaction from a similar yet previously unidentified pathogen. Previous studies conducted in Cambodia have iden- tified the presence of Hantaviruses in rodent populations.28,29 In addition, because of biosafety and biosecurity limitations, we were unable to perform antibody neutralization studies on these samples. The pathogen yield from blood culture was considerably low, likely caused by the single sampling used. However, we were able to detect a number of known pathogens (e.g., B. pseudomallei, S. typhi, S. paratyphi) as well as common skin contaminants (e.g., coagulase negative Staphylococcus, microbacterium). In addition, there were a number of patho- gens that have limited reports of causing disease such as Sphingomonas paucimobilus,30 that require further investiga- tion. Planned studies at these sites will expand molecular and serological techniques to determine the prevalence of bacte- rial pathogens among patients with AFI in Cambodia. Co-detection was limited to PCR-positive results or culture/ smear results where organisms were visualized. This work