Although focal DENV transmission has been noted previously [14
], to our knowledge this is the first study to demonstrate, using control clusters and precise human and entomological data, recent DENV transmission that was focal through space and over a short time span (15 d). DENV-infected hosts (27 enrollees) and vectors (eight Ae. aegypti
) were exclusively identified in the 12 dengue-positive clusters, despite a nearly 1:2 ratio of enrollees between positive and negative clusters. Furthermore, we observed significant central clustering of DENV cases within positive clusters.
We suspect that focal transmission was associated with recent DENV introductions because of the 217 paired serologic specimens from positive cluster enrollees, only one revealed an elevated but declining immunoglobulin M level, which would be indicative of a recent DENV infection occurring up to 60 d prior to cluster initiation [22
]. Consequently, we attributed the observed DENV transmission (enrollees with viremia on day 0 or 15 and/or seroconversion between days 0 and 15) to recent virus introductions. This conclusion is in contrast, however, to data published by Beckett and others [20
] who conducted cluster investigations in West Jakarta, Indonesia. They detected 175 recent DENV infections upon enrollment in 53 positive clusters compared to our one in 12 positive clusters, arguing against recent virus introduction. We attribute these contrasting results to study design differences. First, we recruited from schools whereas Beckett recruited from a hospital, potentially after the virus had undergone significant community-based amplification. Second, we preferentially enrolled children as the primary susceptible and amplifying portion of the host population. Beckett additionally enrolled adults. Adults may have exhibited greater background dengue immunity that may have confounded the serologic data. Third, Beckett's study was conducted in an urban area, in contrast to rural villages in our study. Differences in transmission dynamics between these kinds of habitats were likely shaped by the frequency of DENV introductions and diversity in human behaviors.
Previous studies have documented hyperendemicity of all four DENV serotypes with an approximate 5% annual risk of acquiring an infection in KPP [19
]. In our study, cluster number 4 had a 52% attack rate among enrollees sampled during the 15-d follow-up period. However, after excluding this cluster and its matched negative cluster, the adjusted AR remained high (six per 100). This number represented a 12.4% risk of an enrolled child acquiring a DENV infection within a 15-d period when living within 100 m of a child ill with dengue. Eleven of 12 positive clusters had at least one enrollee with acute dengue in addition to the index case. Given the required intrinsic incubation period, and the finding that all eight virus isolates from mosquitoes matched the serotype recovered from the index case suggest, though not definitively, that except for children from whom virus was recovered on day 15, multiple viremic children within a cluster were infected by one or very few infected mosquitoes. Other evidence within our study to further support village- and not school-based vector sources of DENV infection are that: (1) mosquito populations in schools were extremely low, (2) children seroconverting to dengue within a cluster attended different classrooms within the school, (3) genomic sequences of the envelope (E)-regions of the viruses isolated from children and mosquitoes within the same villages were identical (R.G. Jarman, unpublished data), and (4) housemates of dengue seroconverters had a higher relative risk for DENV infection than those of nondengue seroconverters. The latter observation is consistent with previous reports [14
]. We suspect that the predominance of DENV transmission in KPP villages reflects, at least in part, routine and effective vector control in schools (insecticide every May and July and Temephos to containers every 3 mo), but not in village homes.
Differences in transmission observed between positive and negative clusters could not be attributed to differences in enrollee demographics. Differences in behavioral factors, however, could not be excluded. Within positive clusters, risk of infection decreased with age for males and increased with age for females. This observation merits further investigation with a larger sample and analysis of sex-specific behaviors that might modify risk of infection with advancing age.
The only statistically significant determinant among environmental features associated with focal DENV transmission was the greater availability of piped water in negative clusters. Though one may consider a causal relationship (that is, less piped water availability leading to greater need for water storage leading to more containers for larval mosquito development resulting in higher dengue risk), we found no difference in the number of containers between cluster types. Although accurate data on water turn-over are difficult to obtain, the greater number of positive containers in positive than in negative clusters could not be explained by a difference in the frequency of container turn-over rates that we measured. These data could reflect a historical norm or behavior in response to lack of reliability of piped water possibly guided by people's knowledge of dengue preventive measures [33
The only statistically significant difference among entomological indices was the greater number of Ae. aegypti
pupae per person in positive than negative clusters. It is important to note that observed mean pupae per person exceed by an order of magnitude the minimum entomological threshold estimated by Focks and others [34
] for a different region of Thailand. This implies that even when pupal densities are relatively high, differences in this measure of entomological risk can be epidemiologically informative. Although adult mosquito population density tended to be higher in positive clusters, differences were not statistically significant, perhaps due to limitations in sampling adult Ae. aegypti
with backpack aspirators. Alternatively, mosquito density may be most informative when viewed in concert with herd immunity, and mosquito density alone may be less relevant than the presence of DENV-infected mosquitoes that potentially can transmit virus to multiple individuals [2
]. Dengue cases in enrollees occurred over a wide range of female Ae. aegypti
densities (). At densities higher than approximately 1.5 Ae. aegypti
females per child, clusters were more likely to be positive than negative. This indicates that DENV transmission was more likely to occur at higher vector densities.
Relationship between Vector Density and Dengue Cases
Perifocal spraying is a common approach by health departments to contain/control dengue. However, this practice has been found to be ineffective in aborting DENV transmission [13
]. Our data suggest that if school-based surveillance can be bolstered by rapid, easy-to-use, and affordable diagnostics, spatially and temporally focused vector control in rural areas such as KPP could be more effectively applied to contain new virus introductions and offset the theoretical risk of longitudinal transmission within and beyond village foci. Although the risk of infection decreased significantly with distance from the center of a cluster, we did not examine people living beyond 100 m of an index case. Our study did not define the spatial dimensions of DENV transmission. Nevertheless, we expect that interventions will need to go beyond a 100 m radius of the home of a DENV-infected child because viremic residents or visitors bitten by an infected mosquito can move virus farther than a flying, infected adult female Ae. aegypti
We do not know the longitudinal effects of killing adult mosquitoes on transmission within a community. Koenraadt and others [27
] determined in our study area that within 1 wk of spraying insecticide inside homes, approximately 50% of prespraying levels of Ae. aegypti
populations were reestablished. Identifying only two of 217 child enrollees with dengue viremia on day 15, both approximately 50 m from the index case within the same positive cluster, indicates that vector control can be locally successful when promptly and properly applied in response to a dengue case. Insecticide applications are most effective when applied inside homes where most Ae. aegypti
] and otherwise avoid contact with insecticides applied outdoors [35
Though our study design was rigorous, our conclusions must be considered in the context of largely logistical limitations: (1) We did not sample all children and mosquitoes within the cluster area. (2) We were unable to characterize the serotype of all DENV infections among village enrollees given restrictions in the frequency of collecting blood from children. (3) We did not collect data on human mobility/behavior that may have influenced the dynamics of transmission within the villages. (4) The possible contribution of adults to DENV transmission was not studied. (5) We did not study the seroprevalence profiles of cluster enrollees. Future studies should focus on positive clusters to more fully characterize the transmission dynamics, the impact of human behavior on transmission patterns, the appropriate spatial scale for disease surveillance/control, and identify more practical and cost-effective approaches to rapid dengue diagnosis.
Our cluster methodology provided additional epidemiologic insights. Of note, 14 of the 27 cases of dengue among enrollees were clinically inapparent during this period when DENV-4 was the primary serotype circulating. Most (five of six) primary DENV infections detected in our study were clinically inapparent, similar to observations during a predominantly DENV-2 transmission year in Bangkok [38
]. The nearly 1:1 ratio of inapparent to symptomatic secondary DENV infections in our study is also consistent with previous results from KPP [19
]. DHF occurred in one (8%) of 12 symptomatic infections and one (4%) of 27 DENV infections confirming that severe dengue represents only a small fraction of the total DENV burden. Future cluster studies can complement these clinical and virologic data by examining correlates of protection that limit transmission, early immunologic events via postinoculation pre-illness specimens and their association with disease severity and sequence variation among viruses through time and space as they circulate between human and mosquito hosts.
The prospective cluster methodology utilized here and by others [20
] has the potential for broad application. It can be used for multidisciplinary transmission studies of other vector-borne viral diseases as well as spatially and temporally clustered infectious diseases.