Assessments of transmission patterns and true incidences of dengue or DENV-infections are resource intensive and have been performed in a limited number of settings. Multi-year studies with active, community-based surveillance for febrile illness have been conducted in South East Asia (Kamphaeng Phet 
and Ratchaburi, Thailand 
, A Sabchareon, personal communication, unpublished data); Long Xuen, Viet Nam 
and Bandung, West Java, Indonesia 
and in the Americas (Iquitos, Peru 
and Managua, Nicaragua 
). Only one study determined the incidence of dengue among adults (18–66 year-old factory workers) in Indonesia 
. All the others determined disease incidence in children using various active surveillance methods to detect febrile illness, including school absenteeism and self-reporting by participants. All studies included clinical follow-up to identify patients for serologic and molecular testing for DENV infection. Similar to our study, surveillance was mainly conducted during the dengue season in Kamphaeng Phet Thailand), while sites in Ratchaburi, Thailand, Vietnam, Nicaragua, Peru and Indonesia conducted year-round surveillance. However, all these studies only conducted surveillance in relatively populated urban or semi-urban areas. In addition, community-based active surveillance for febrile illness in all age groups was conducted during several large dengue epidemics in rural and urban areas of Cuba in 1997 and 2001 
although only surveillance data from urban areas were published.
Although there were some methodological differences in how active surveillance was conducted in our study and those identified above (e.g., house-to-house visits compared to school absenteeism or self-identification of fever episodes), we found annual disease incidence rates of 1.3–5.7% over three years, which was higher than those observed in Kamphaeng Phet (0.8–3.6% in 4–14 year-olds) or Ratchaburi, Thailand (1.6% in 4–14 year-olds) (26), Nicaragua (0.4–1.9% in 2–11 year-olds), Vietnam (1.7–4.0% among 2–15 year-olds) or Peru (1.0% in 5–17 year-olds). Moreover our highest incidence rates were among pre-school children ages compared to Thailand and the Americas where the highest age-specific incidence rates were among children >8–9 years of age 
For several reasons the incidence observed in our study may still have underestimated the true disease burden although our use of house-to-house case finding was most likely to identify febrile children in a low-literacy rate population where attendance at public health centers is thought to be low 
. First, surveillance was not performed throughout the year and dengue cases are known to occur at low rates between dengue seasons. However, based on nationally reported cases, those that occur during the non-dengue season only account for <5% of the total 
. Second, in the first year of the study (2006) our case definition required a fever ≥48 hrs and only a third of children with a febrile episode met those criteria. However it is unlikely that a significant number of cases were excluded since in subsequent years <4% of dengue cases had fever duration <2 days. Third, in 2007 a large dengue epidemic occurred in the country resulting in ~4 fold increase of cases reported to NDCP as compared to the previous 5 years 
. Since the NDCP data indicated that the epidemic in 2007 started in Cambodia approximately one month before our surveillance began and accounted for ~25% of the total reported cases 
; we may have missed this proportion of cases prior to our start of active fever surveillance in June. Finally, our laboratory diagnostic algorithm only tested acute phase samples from patients with fever when the convalescent sample tested positive for anti-DENV IgM. In a setting such as ours where there is a high frequency of secondary DENV infections, up to 28% of secondary infections might not be detected serologically because of low IgM levels 
Dengue is highly focal in its geographic distribution and this was observed during this surveillance study. Dengue incidences by village showed wide ranges and a markedly diverse spatial distribution within the study area and population. DENV-3 predominated in 2006 in Cambodia and was reported in 16 of 24 provinces in Cambodia (Ministry of Health's unpublished data). However, during the same year an outbreak in one of the participating villages was caused by DENV-1 and was responsible for two-thirds of all identified cases in the study area. In 2008, one village was affected by DENV-2 with an attack rate of ~13% and accounted for 34% of all identified cases. A wide range of incidences among the villages and urban areas was also observed during the 2007-epidemic.
Another finding in this surveillance study was that rural areas were usually affected by dengue to the same degree as urban areas or, as during the 2007-epidemic, at even higher attack rates. Factors thought to explain the perceived urban predominance of dengue included rapid human population growth, the presence of an unreliable or absent water supply which forced residents to store water in open containers and the presence of other man-made containers, all of which serve as major production sites for Aedes aegypti 
All of these conditions are present in rural areas of Cambodia as the country develops without proper water supplies and persons from rural areas frequently travel between large cities and relatives in their villages. The villages in our surveillance study were located close to one of the main national roads with constant daily traffic between villages and Phnom Penh and the opportunity for dengue introduction from viremic individuals passing through the area. The expansion of dengue to rural areas has also been observed in Thailand and India 
. In the absence of a dengue vaccine, this finding underscores the importance of conducting education and vector control programs in rural as well as urban areas of Cambodia. The high dengue incidence in children <7 years of age who rarely leave the villages and are not yet in school, argues for ongoing transmission within Cambodian villages and around households as has been recently observed in Thailand 
Assuming that hospitalization was a surrogate for disease severity, 2006 appeared to have been particularly severe with 46% of dengue cases hospitalized. However, our study area may not have been representative of the country. A DENV-1-related outbreak occurred primarily in one village in the study area and resulted in a higher hospitalization frequency among younger children, while the predominant virus circulating in the country was DENV-3. Because DENV-1 has circulated less frequently than DENV-2 or DENV-3 in Cambodia 
, it may have caused more severe disease expression among a largely susceptible population. In subsequent years, DENV-4 and DENV-2 appeared to have been associated with milder disease than DENV -1 and DENV-3. Less severe disease expression has been previously described for DENV-4, however, milder expression of DENV-2 dengue came as a surprise since this serotype was associated in previous studies from Thailand with increased disease severity 
. Nevertheless, clinical expression may also depend on genetic sub-strains within the four serotypes, pre-existing antibody to DENV or the sequence of infections with other serotypes 
. This phenomenon of apparent attenuation of dengue disease severity at a population level was also seen observed after the large epidemics of 1998 in the Philippines and Thailand 
. We were limited in further analyses since circulation of serotypes was year-dependent: only 5% of DENV-2 detected in this study circulated in 2006 and 2007 and no DENV-1 was identified in 2008.
Our surveillance study highlights once again the high degree of under-recognition and under-reporting of this disease and the variation in dengue epidemiology which occurs annually because of the cyclical variation in incidence. A number of factors are thought to influence DENV transmission and disease expression such as environment 
, human and vector behaviors 
, possible virulence of the virus strains 
, and the human host 
(baseline immunity and cross-protective immunity). Given the cyclical nature of dengue incidence with broad variations from one year to another, we underscore the importance of maintaining long term population-based surveillance sites to better estimate true incidence and define the dynamics of dengue epidemiology and serotype circulation. Long-term epidemiological data on dengue and DENV transmission obtained though population-based surveillance studies will also be needed to for evaluation of promising dengue vaccine candidates both through randomized, controlled clinical trials of efficacy 
and studies of vaccine effectiveness.