Beyond efficacy in water containers: Temephos and household entomological indices in six studies between 2005 and 2013 in Managua, Nicaragua

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Abstract

Background

A cluster-randomized controlled trial of community mobilisation for dengue prevention in Mexico and Nicaragua reported, as a secondary finding, a higher risk of dengue virus infection in households where inspectors found temephos in water containers. Data from control sites in the preceding pilot study and the Nicaragua trial arm provided six time points (2005, 2006, 2007 and 2011, 2012, 2013) to examine potentially protective effects of temephos on entomological indices under every day conditions of the national vector control programme.

Methods

Three household entomological indicators for Aedes aegypti breeding were Household Index, Households with pupae, and Pupae per Person. The primary exposure indicator at the six time points was temephos identified physically during the entomological inspection. A stricter criterion for exposure at four time points included households reporting temephos application during the last 30 days and temephos found on inspection. Using generalized linear mixed modelling with cluster as a random effect and temephos as a potential fixed effect, at each time point we examined possible determinants of lower entomological indicators.

Results

Between 2005 and 2013, temephos exposure was not significantly associated with a reduction in any of the three entomological indices, whether or not the exposure indicator included timing of temephos application. In six of 18 multivariate models at the six time points, temephos exposure was associated with higher entomological indices; in these models, we could exclude any protective effect of temephos with 95% confidence.

Conclusion

Our failure to demonstrate a significant protective association between temephos and entomological indices might be explained by several factors. These include ecological adaptability of the vector, resistance of Aedes to the pesticide, operational deficiencies of vector control programme, or a decrease in preventive actions by households resulting from a false sense of protection fostered by the centralized government programme using chemical agents. Whatever the explanation, the implication is that temephos affords less protection under routine field conditions than expected from its efficacy under experimental conditions.

Trial registration

ISRCTN 27581154.

Electronic supplementary material

The online version of this article (doi:10.1186/s12889-017-4296-6) contains supplementary material, which is available to authorized users.

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Most cited references 17

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Community involvement in dengue vector control: cluster randomised trial

V Vanlerberghe, M E Toledo, Benjamín M Rodríguez … (2009)

Objective To assess the effectiveness of an integrated community based environmental management strategy to control Aedes aegypti, the vector of dengue, compared with a routine strategy. Design Cluster randomised trial. Setting Guantanamo, Cuba. Participants 32 circumscriptions (around 2000 inhabitants each). Interventions The circumscriptions were randomly allocated to control clusters (n=16) comprising routine Aedes control programme (entomological surveillance, source reduction, selective adulticiding, and health education) and to intervention clusters (n=16) comprising the routine Aedes control programme combined with a community based environmental management approach. Main outcome measures The primary outcome was levels of Aedes infestation: house index (number of houses positive for at least one container with immature stages of Ae aegypti per 100 inspected houses), Breteau index (number of containers positive for immature stages of Ae aegypti per 100 inspected houses), and the pupae per inhabitant statistic (number of Ae aegypti pupae per inhabitant). Results All clusters were subjected to the intended intervention; all completed the study protocol up to February 2006 and all were included in the analysis. At baseline the Aedes infestation levels were comparable between intervention and control clusters: house index 0.25% v 0.20%, pupae per inhabitant 0.44×10−3 v 0.29×10−3. At the end of the intervention these indices were significantly lower in the intervention clusters: rate ratio for house indices 0.49 (95% confidence interval 0.27 to 0.88) and rate ratio for pupae per inhabitant 0.27 (0.09 to 0.76). Conclusion A community based environmental management embedded in a routine control programme was effective at reducing levels of Aedes infestation. Trial registration Current Controlled Trials ISRCTN88405796.

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Oviposition Site Selection by the Dengue Vector Aedes aegypti and Its Implications for Dengue Control

Jacklyn Wong, Steven Stoddard, Helvio Astete … (2011)

Introduction Dengue viruses are transmitted to humans primarily by the mosquito Aedes aegypti and represent an increasing public health concern in tropical and subtropical regions worldwide. Because no vaccine or antiviral therapy is commercially available, controlling the mosquito vector is the only current means to prevent dengue outbreaks [1]. Contemporary control campaigns, rather than attempting to eradicate Ae. aegypti, aim to suppress mosquito populations below a threshold density at which they no longer support viral amplification [2]. Controlling adult mosquitoes is made challenging by the behavior of domestic Ae. aegypti. Adult Ae. aegypti rest inside homes, typically on clothing, curtains, bedspreads, and furniture, items that cannot be sprayed with residual insecticides [3]. Aerosol space sprays consist of small airborne droplets of insecticide designed to kill adult mosquitoes on contact, but difficulty in reaching indoor adult resting sites can limit their efficacy [4]. Even when space sprays are effective in reducing adult populations, effects are transient due to the continuing emergence of new adults or immigration from untreated areas [3], [5]. Insecticide-treated materials (curtains, water container covers, and bednets) have shown promise in reducing Ae. aegypti populations [6], [7], but the impact of these reductions on dengue transmission has not been determined. Currently, the World Health Organization recommends directing routine Ae. aegypti control toward the immature stages [2]. Ae. aegypti females lay eggs singly just above the water line, often in man-made containers located in the home or yard (buckets, drums, tires, and vases, etc.) [8]–[10]. Eggs hatch when inundated, and larvae develop by filter feeding and browsing for microorganisms and organic detritus [11], [12]. Control approaches such as container removal (source reduction) and larvicide application aim to reduce the number of new emerging adults in the population [2]. Traditional models of Ae. aegypti assume that population dynamics are regulated predominantly by density-dependent competition for food during early larval stages and little affected by oviposition rates [13], [14]. Based on these models, some researchers have assumed that all containers suitable for larval development receive an excess of eggs, thereby leading all larvae to experience density-dependent competition [15]. This is the rationale behind targeted source reduction (a WHO-recommended control strategy) and the expectation that eliminating containers that, for example, produce 75% of adults will lead to a proportionate decrease in the overall adult population [15]–[17]. Much remains unclear, however, about the factors regulating Ae. aegypti adult production and how reducing, but not eliminating, containers will ultimately affect adult abundance. In some mosquito species, female choice in oviposition site is adaptive and can influence population distribution and dynamics [18], [19]. Females can enhance survival and development of their offspring by selecting egg-laying sites that reduce exposure to predators and competitors [19], [20], or increase access to food [21], [22]. In general, understanding insect egg-laying decisions may provide additional insight into the factors affecting population regulation and aid in predicting how populations will respond to control measures [23]. Oviposition preferences by Ae. aegypti have been studied in the laboratory [24]–[31], but to a lesser extent in the field [32]–[36]. Research has typically involved varying one or two oviposition site factors at a time and observing the number of eggs laid in response (reviewed in [24]). Such studies reveal the types of abiotic and biotic stimuli potentially affecting oviposition, but yield limited information on the relative importance of these stimuli in nature [24], [37]. The goals of our study were to test whether free-ranging Ae. aegypti females make active choices regarding where they oviposit and to identify factors influencing oviposition. Although selective oviposition has been demonstrated using small oviposition traps in the field [32]–[35] or water-storage containers in an enclosure [36], we examined for the first time females' oviposition choices among naturally-occurring containers in homes throughout a large, dengue-endemic city. We also investigated the consequences of oviposition site selection for offspring fitness by testing whether females choose sites to maximize the amount of food available for their progeny. Food availability is known to affect components of mosquito fitness such as offspring survival, development time, and adult size [38]. Lastly, we considered the implications of selective oviposition behavior for Ae. aegypti population regulation and the success of targeted larval control strategies. Materials and Methods Study location Our study was conducted in Iquitos (73.2°W, 3.7°S, 120 m above sea level), a city of approximately 380,000 people located in the Amazon Basin, Department of Loreto, Northeastern Peru [10], [39]–[41]. Rain falls during all months of the year and average temperature and relative humidity are fairly consistent [42]. During our study period from July 2007 to August 2009, mean monthly temperature ranged from 24.8°C (±1.1 SD) in June 2008 to 26.5°C (±1.1 SD) in December 2008. Average relative humidity ranged from 80.2% (±4.1 SD) in August 2007 to 86.2% (±4.4 SD) in April 2009. More detailed climate data for the years 2007 to 2009 are given in the Supporting Information (Table S1). In response to the unreliable municipal water supply, Iquitos residents store water in containers [40]. Household containers are filled in three primary ways: 1) from spigots in the home or neighborhood (manually filled), 2) intentionally placed outside to collect rain water (rain-filled), and 3) filled with rain water as a result of being untended outside (unmanaged). Method of filling is correlated with the frequency of water turnover and amount of organic detritus present in containers, with manually filled containers kept the cleanest and unmanaged containers collecting the most organic material. Containers in Iquitos generally lack predators of larval Ae. aegypti, such as copepods or fish (ACM and JW, unpublished data), but do occasionally contain immature Culex which may act as competitors [10]. Ae. aegypti are reproductively active all year in Iquitos. Of the roughly 290,000 containers examined by Morrison et al. [10], 7.3% contained Ae. aegypti larvae and/or pupae. Observational study Consent process The households included in this study were identified through three ongoing, longitudinal cohort studies on dengue transmission dynamics approved by the University of California, Davis (Protocol #2006.14381, 2006.14405, 2007.15244) and Naval Medical Research Center Detachment (Protocol #NMRCD 2007.001, NMRC 2005.0009, NMRCD2007.007) Institutional Review Boards (IRBs). As described in detail by Morrison et al. [43], Ae. aegypti abundance surveys were conducted in private homes by two-person teams that administered a brief questionnaire to residents, counted the number of water-holding containers present on the property, inspected containers for immature Ae. aegypti, and collected adult mosquitoes using backpack aspirators. Entomological surveys required a verbal informed consent process in which the survey procedures were explained to residents and if they consented, the survey team was allowed into the household. Both IRBs approved verbal consent without written documentation because the survey form would indicate consent of the residents. Our oviposition study was approved by the local ministry of health (Dirección Regional de Salud -Loreto). The Naval Medical Research Center IRB determined that our study (Project #: PJT-NMRCD.032) did not meet the definition of human subject research. Survey procedures We conducted a large-scale survey to examine female oviposition choices among naturally-occurring containers in Iquitos homes. For nine weeks during July to September 2007 (collection period 1), seven weeks during May to July 2008 (collection period 3), and six weeks during October to December 2008 (collection period 4), we closely observed the number of Ae. aegypti eggs laid in containers within a subset of surveyed houses. Collection period 2 is described later. Each week, 3 to 6 houses having at least one Ae. aegypti-positive container were selected to be included in this study. For each of those houses, we visited 2 to 3 additional houses on the same block (matched in time and space) that had containers but no larvae, such that 9 to 18 total houses were visited per week. All surveyed houses, along with their associated entomological data, were geocoded using a geographic information system previously developed for Iquitos [39]. In each selected house, 2 to 4 inspectors examined the entire property (indoors and outdoors) for water-filled containers and used strips of brown paper towel to line the inside of containers (limited by homeowner permission) at the water line to collect eggs. The following characteristics were recorded on the first day: container size (circumference, capacity, and water volume), location and sun exposure, lid presence, fill method, insecticide treatment, conspecific larvae (abundance and estimated mean density), presence of conspecific pupae, and presence of immature Culex (Table 1). Insecticide treatment (temephos or pyriproxyfen) was scored depending on whether an insecticide sachet was present in containers; we did not determine how long sachets had been in containers or whether insecticidal activity was still active. The abundance of larval Ae. aegypti and the presence of larval Culex were noted by visual inspection without removing larvae. Larval Ae. aegypti estimates per container were categorized as: none, 1 to10, 11 to 50, 51 to 100, or >100 larvae. Estimated mean density of Ae. aegypti larvae was calculated by dividing the midpoint of the larval abundance category (or 200 in the case of >100 larvae) by water volume. Any pupae occurring in containers were collected daily and brought to the field laboratory to be counted and the emerging adults identified as either Ae. aegypti or Culex spp. If third instar Ae. aegypti larvae (determined by size and morphology) were present on the first day, up to 25 third instars were removed per container for starvation bioassays (described below) to assess food availability in containers [38], [44]. Otherwise, mosquito larvae were left undisturbed. 10.1371/journal.pntd.0001015.t001 Table 1 Container characteristics recorded during oviposition survey in Iquitos, Peru, and regression parameters for oviposition models. Variable Levels No. containers Median (range) Circumference Continuous 126 cm (10; 540) (Circumference)2 Continuous 15,791 cm2 (100; 291,600) Location and sun exposure Indoor (enclosed by roof and at least 3 walls) 134 Outdoor shade (exposed to sunlight 100 larvae, retained during survey 45 >100 larvae, removed on day 1 17 Ae.aegypti pupae Absent 454 Present 137 Immature Culex Absent 560 Present 31 Collection period 1 - July to September 2007 (9 weeks) 222 2 - January to May 2008 (14 weeks) 202 3 - May to July 2008 (7 weeks) 93 4 - October to December 2008 (6 weeks) 74 Paper strips were checked daily for three consecutive days to collect a representative sample of eggs laid within each house. Collections were conducted between 09:00 to 12:00 h to minimize disturbance of ovipositing females [45]. If eggs were present, new paper lining was exchanged. Papers with eggs were brought to the field laboratory to count eggs under a dissecting microscope at 20× magnification. Subsamples of collected eggs were hatched once a week to confirm their identity as Ae. aegypti. To prevent production of adult mosquitoes in sampled houses, containers with larvae were overturned or treated with pyriproxyfen at the conclusion of the 3-day survey. During 14 weeks from January to May 2008 (collection period 2), we surveyed containers following the above procedures, with the exception that all larvae and pupae were removed using a net and/or turkey baster on the first day. Therefore, no immature mosquitoes were present in containers when females oviposited on the following three days, but the water was “conditioned” by the previous presence of immatures. All larvae were taken to the field laboratory, where they were enumerated to genus and instar. Up to 25 third instar Ae. aegypti per container were used for starvation bioassays as described below. Data analysis Regression analyses were conducted using R version 2.8.1 [46]. To check for spatial autocorrelation among containers surveyed in the same week as a potential confounder, we estimated Moran's I for egg counts using a Euclidean distance matrix with the APE package within R [47]. Because no spatial structure was evident, subsequent analyses did not take spatial coordinates into account. We attempted to include the density of adult female Ae. aegypti as a predictor variable in our models, but collections were too sparse (mean = 0.14±0.52 SD females per house) for meaningful analyses. Instead, using a separate chi square test, we examined whether the presence of Ae. aegypti larvae was independent from capture of adult females during the abundance survey. To identify variables that best predicted whether or not female Ae. aegypti laid eggs in a container, a logistic regression model was fitted to our data (1 = container received eggs at least once during three days of observation, 0 = container received no eggs). Categories of Ae. aegypti larval abundance were further divided depending on whether larvae were retained during the survey or removed from containers on the first day. Collection period was included to control for time. The three measures of container size were collinear (circumference-capacity, Spearman's ρ = 0.86; circumference-water volume, Spearman's ρ = 0.65, capacity-water volume, Spearman's ρ = 0.85). Because the amount of space available for oviposition is determined by container circumference, we included circumference rather than capacity or water volume in our model. Larval abundance and estimated mean larval density also were collinear (Spearman's ρ = 0.92). Larval abundance was used because it provided a better model fit to the data. Starting with a saturated model including all variables listed in Table 1, we employed a log-likelihood test to eliminate, stepwise, the non-significant variable with the greatest χ2 p-value (2× log-likelihood of current model–2× log-likelihood of previous model ∼χ2, df = 1, p>0.10). If the final model included a variable with more than two levels, Tukey's multiple comparisons were applied using the MULTCOMP package [48] to identify differences in level effects. Only containers receiving eggs were included in the analysis to identify variables influencing the number of eggs laid in containers. Negative binomial regression was performed using the MASS package [49]. Our response variable was the mean number of eggs laid per container per day, rounded to the nearest integer. To more closely examine the association between egg abundance and container size, we included both container circumference and (circumference)2 as predictor variables in the model. As with the logistic regression model, containers were classified according to larval abundance and whether or not larvae were removed on the first day, and to collection period to control for time. Model selection was based on the log-likelihood test. To confirm that model assumptions were met, deviance residuals were plotted against: (1) fitted values, (2) each explanatory variable included in the model, (3) each explanatory variable eliminated from the model, (4) survey date, and (5) spatial coordinates [50]. Starvation bioassays We measured larval resistance to starvation (RS, number of days larvae survive without food) as an indirect measure of per capita food availability in containers [38]. In general, mosquito larvae that consume more food are able to store more energy reserves and resist starvation longer [13], [44]. During the above-described survey of Iquitos containers, 5 to 25 third instar larvae were removed from containers in the field and transferred to individual plastic cups (5 cm diameter×6 cm height) filled to 2/3 capacity with bottled drinking water. Third instars were used for bioassays because fourth instar Ae. aegypti frequently pupate when starved [44]. Cups were placed indoors in our field laboratory, where larvae were exposed to natural light and temperature. Water was changed every two days to prevent accumulation of waste and microbial growth [51]. Time to death (in days) was recorded for each larva. Because starvation times were not distributed symmetrically for larvae from each container, the median larval RS was used as the measure of central tendency for the data for each container. Spearman rank correlation was used to identify any association between larval RS and egg density (mean eggs laid per day/circumference). Data were stratified according to whether or not all larvae had been removed from containers on the first survey day. To account for potential effects of larval abundance and container size, data also were stratified by larval abundance (≤50 larvae vs. >50 larvae) and container capacity (≤20 L vs. >20 L). Experimental study For 12 weeks during June to August 2009, we carried out an experimental study manipulating both the presence of conspecific larvae and accumulation of organic material in containers and recorded oviposition by wild females. This experiment was replicated in three central Iquitos residences, the courtyard of our field laboratory and in the yards of two other houses selected based on the consistent presence of Ae. aegypti and homeowner willingness to participate. At each residence, three identical 6-liter blue plastic buckets (20 cm diameter×23 cm height) were placed close to one another (0.5 m apart) to minimize differences in container position. Hourly at each house, ambient temperature and relative humidity were recorded using a Hobo® ProV2 data logger (U23-001; Onset Computer Corporation, Pocasset, MA) and water temperature was recorded in one container per house using a Hobo® Pendant logger (UA-002-64). We created three container treatments: A (unmanaged, with larvae), B (unmanaged, no larvae), and C (manually filled, no larvae). Unmanaged containers (A and B) were filled with four liters of tap water and allowed to accumulate organic debris for 12 weeks, whereas manually filled containers (C) were cleaned and refilled with new tap water every other day. Fifty first instar Ae. aegypti larvae were introduced into treatment A containers every two weeks starting on the first day. Oviposition was monitored by lining the inside of buckets with strips of brown paper towel to collect eggs. Every second day, papers were exchanged and the number of eggs counted as described above. On egg collection days, we also temporarily removed larvae from treatment A containers to determine their developmental stage and count them. Larvae were then returned to the container from which they originated. To estimate the accumulation of organic detritus and bacterial growth in unmanaged containers, a thoroughly mixed water sample was measured for cloudiness using a turbidity tube [52] and dissolved oxygen content using an Ecological Test Kit (Rickly Hydrological Company, Columbus, OH). Water samples were returned to containers after testing. In all containers, tap water was added every few days to replace water lost to evaporation. Any pupae were removed to prevent emergence of adult Ae. aegypti. Data analysis Due to repetitive sampling, effects of treatment (A, B, or C), house, and week on the number of eggs laid per week ( transformed) were analyzed by repeated measures analysis of variance (RM ANOVA). RM ANOVA was also used to examine effects of treatment, week, and house on water turbidity ( transformed) and dissolved oxygen content (χ3 transformed). RM ANOVAs were carried out using PROC MIXED in SAS version 9.2 [53] and transformations were performed to meet ANOVA assumptions. Results Observational study We monitored oviposition in 591 containers in 448 households across Iquitos. Ae. aegypti eggs were deposited in 51.8% of surveyed containers (306 of 591). Egg counts per container per day were strongly skewed, with the majority of containers receiving 0 to 50 eggs (median = 2, mean = 41), and a few containers receiving hundreds of eggs (Figure 1). All mosquitoes reared from collected eggs were Ae. aegypti, which we found to be the only Aedes species present in domestic containers throughout Iquitos. The presence of Ae. aegypti larvae in households was independent from whether or not adult females were caught during entomological surveys (χ2 = 1.897, df = 1, p = 0.169). Culex mosquitoes were occasionally present in the same containers (5.2% of all containers surveyed, 11.3% of Ae. aegypti-positive containers), but were easily distinguished by morphology. We did not find any containers colonized only by Culex. 10.1371/journal.pntd.0001015.g001 Figure 1 Frequency distribution of Ae. aegypti eggs. Number of eggs collected per day in naturally-occurring containers throughout Iquitos, Peru (n = 591 containers). After controlling for collection period, three variables were significant predictors of whether females laid eggs in containers: Ae. aegypti larvae, exposure to sunlight (≥20% of day), and absence of a container lid (Table 2). The probability of oviposition increased when sites held conspecific larvae (β = 1.658; 95% CI = [1.286, 2.030]; p z Intercept −0.379 0.247 −1.533 0.125 Larvae (1–10, retained) 1.285a 0.362 3.546 100, retained) 2.332a 0.470 4.964 100, removed) 1.581a 0.557 2.840 0.005 Location (inside) −0.601b 0.249 −2.416 0.016 Location (outside, shade) −0.538b 0.211 −2.550 0.011 Lid (present) −0.706 0.369 −1.914 0.056 Collection period 2 −0.788c 0.275 −2.863 0.004 Collection period 3 −0.209 0.280 −0.749 0.454 Collection period 4 −1.027c 0.309 −3.327 0.10). Larvae refers to Ae. aegypti. Parameter estimates followed by the same letter are not statistically different from one another as indicated by Tukey's multiple comparisons. Significant p-values are indicated in bold. Among containers receiving eggs, the number of eggs laid was affected by larval abundance, whether larvae were removed prior to oviposition, pupae, fill method, circumference, and (circumference)2 (Table 3). Females laid more eggs when over 50 conspecific larvae were present in containers (β = 0.759; 95% CI = [0.483, 1.035]; p z Intercept 2.964 0.311 9.543 100, retained) 0.784a 0.227 3.459 100, removed) 0.838a 0.363 2.308 0.021 Pupae (present) 0.448 0.156 2.864 0.004 Fill method (manual) 0.073 0.160 0.458 0.647 Fill method (unmanaged) 0.387 0.150 2.578 0.010 Collection period 2 −0.470b 0.219 −2.144 0.032 Collection period 3 −0.531b 0.169 −3.138 0.002 Collection period 4 −1.293 0.225 −5.754 0.10). Larvae and pupae refer to Ae. aegypti. Parameter estimates followed by the same letter are not statistically different from one another as indicated by Tukey's multiple comparisons. Significant p-values are indicated in bold. Starvation bioassays Third instar larvae were collected for starvation bioassays from 113 containers. For the majority of containers, median larval RS was between 5 to 15 days (range 0 to 28 days) (Figure 3). There were no significant correlations between median RS and the mean density of eggs laid per day (all other larvae retained, n = 59 containers, Spearman's ρ = 0.15; all other larvae removed, n = 54 containers, Spearman's ρ = 0.0008). No correlations were evident when the data were also stratified by larval abundance or container capacity (data not shown). 10.1371/journal.pntd.0001015.g003 Figure 3 Median resistance to starvation vs. average number of eggs laid. Each circle represents an individual container (n = 113 containers). Median resistance to starvation is the median number of days that larvae from a container survive without food. Number of eggs laid in that container was averaged over the three day survey period and divided by container circumference (mm). Experimental study Ambient temperature and relative humidity were measured for the first four weeks and were consistent among the three study locations (field laboratory, house 1, and house 2) (Table 4). Water temperature (Table 4) was recorded for eight weeks and found to be similar for the field laboratory and house 1. Due to logger malfunction, water temperature was not recorded at house 2. Because Iquitos climate was relatively consistent during June to August 2009 (Table S1), we expect the data recorded at each location to be indicative of the entire study period. 10.1371/journal.pntd.0001015.t004 Table 4 Air temperature, relative humidity, and water temperature at three experimental study locations. Air temperature °C (± SD) Water temperature °C (± SD) Location Min Mean Max Mean RH % (± SD) Min Mean Max Field house 24.4±0.7 26.3±0.8 29.2±1.3 82.7±2.5 23.8±0.7 25.4±0.7 27.6±1.2 House 1 24.2±0.8 26.7±1.0 30.9±2.5 82.3±3.0 23.7±0.7 25.3±0.8 27.3±1.2 House 2 23.5±0.7 26.2±0.9 30.6±1.6 84.8±2.6 * * * *Water temperature data are missing from House 2 due to logger malfunction. Conspecific larvae were present in treatment A containers and absent from treatment B and C containers throughout the experiment. The number of Ae. aegypti eggs laid in each container per week was influenced by container treatment (ANOVA F = 77.70; df = 2, 4; p 50% of the day (ACM, unpublished data), in contrast to Iquitos, where abundant tree coverage limits sun exposure to only 10–40% of the day for most outdoor containers. We cannot directly compare our data to that of Barrera et al. [76] because metrics were not provided for container categories of “full sun,” “partial sun,” or “shaded.” We were not able to measure water temperature in each surveyed container. Maximum daily water temperatures from our experimental containers were typically 27–28°C, suggesting that water temperatures are lower in Iquitos compared to Puerto Rico. Attraction to large oviposition sites has been demonstrated in Ae. aegypti [36] as well as other mosquito species [67], possibly because large sites collect more food or are resistant to desiccation. We found that the number of Ae. aegypti eggs laid increased with container circumference up to a threshold around 270 cm, after which oviposition leveled off, indicating that perhaps the relative advantage of large container size diminishes as containers become bigger. Due to the low occurrence in Iquitos of containers exceeding 270 cm in circumference (n = 26 of 591 containers, 4% of surveyed containers), we could not assess the relationship further between increasing container size and oviposition. A major limitation of our study design was the inability to examine effects of container material and/or texture on oviposition. Container texture affects Ae. aegypti oviposition, with females preferring to lay their eggs on rough surfaces [34], [37]. Because we lined containers with strips of paper towel to transport eggs back to the field laboratory, we artificially made container surfaces homogeneous. In a previous Iquitos field study, we showed that females laid more eggs in cement containers compared to plastic or metal containers when all were unlined and similar in size [45]. Additional experimental studies should be conducted to investigate the importance of container material to oviposition site choice when conspecific presence and abundance, fill method, sun exposure, and container size are varied. Ae. aegypti oviposition site choice appears to be flexible, potentially reflecting a mix of site selection strategies across the population. A small portion of females may act as “founders” (e.g., [77]), choosing non-colonized sites based on environmental indicators of quality, whereas the majority of females respond predominantly to conspecific cues. Alternatively, each female may partition her egg batch so that most eggs are laid in colonized containers, when colonized containers are available, and a smaller fraction elsewhere. It should be noted that these scenarios are not mutually exclusive; for any female, the decision to reject or accept a particular site may change with time. For example, results from studies on herbivorous insects demonstrated that ovipositing females typically become more accepting of low-ranking sites as search time progresses (reviewed in [78]). Recent theoretical work on animal decision rules suggests that when individuals are limited by time, number of options, and accuracy with which they can assess site quality, decisions should be based on the best-of-n rule [79]. If female Ae. aegypti use this rule, they are likely to assess a fixed number of sites (n) and choose the perceived best among them, rather than searching longer for a site that meets specific criteria. Such a rule could explain the oviposition patterns we observed in Iquitos; colonized containers tend to be utilized when found, but other site characteristics (size, sunlight, and organic detritus) are used to judge site quality if the n sites do not include a colonized container. This remains to be confirmed in the field. Decision rules used by Ae. aegypti to select oviposition sites merit further investigation. Female choice of oviposition site may have greater impact on Ae. aegypti population dynamics than previously thought. We propose that, due to strong conspecific attraction, oviposition site selection could lead to dense aggregations of larvae and actually contribute to density-dependent regulation. This phenomenon may explain why larvae in the field frequently develop under food-limiting conditions [9], [38], [80]. It is likely that while some colonized sites become crowded, other suitable larval development sites remain empty. A companion study in Iquitos indicated comparable survival and development rates when larvae were reared in water collected from colonized vs. non-colonized containers in the field, suggesting no difference in food content (STS, unpublished data). These results imply that availability of larval food is not the primary determinant of oviposition choices and agree well with our larval starvation data presented herein. A similar study in Trinidad, West Indies, revealed no difference in nutrient levels between water-storage drums colonized or not by Ae. aegypti [81]. Our results have direct implications for strategies to control Ae. aegypti. Targeting containers that produce the most Ae. aegypti adults for removal or larvicide treatment will reduce mosquito populations in the short term. Sustained population suppression, however, will be difficult to achieve by these means. Elimination of highly productive containers (or the immature Ae. aegypti within) will likely shift new eggs to alternative suitable containers. If immature conspecifics are no longer available as a strong oviposition cue, females that would have concentrated their eggs in those highly productive sites may instead oviposit among suitable, previously unoccupied containers based on food availability and/or sun exposure. Strategies that kill mosquitoes late in their development (i.e., insect growth regulators (IGRs) that kill pupae [82], [83] rather than larvae) will enhance vector control by creating “egg sinks,” treated containers that exploit conspecific attraction of ovipositing females, but reduce emergence of adult mosquitoes via density-dependent larval competition and late acting insecticide. For an egg sink strategy, it would be best to employ IGRs that have no repellent effects on ovipositing females, such as pyriproxyfen [84] or methoprene [85]. Pyriproxyfen is of particular interest because adult females are able to transfer the IGR to other oviposition sites [84], [86]. Thus, pyriproxyfen-treated containers could potentially serve as both egg sinks and sources for insecticide dissemination. The success of this approach would depend on oviposition patterns of individual females. Alternatively, rather than relying on conspecific larvae, control tools could be designed to capitalize on the attractant or stimulant properties of semiochemicals influencing Ae. aegypti oviposition responses in the field. Bacteria-derived oviposition attractants could be used to lure females to lethal ovitraps or stimulants could be used to increase their exposure to insecticide-impregnated substrates [30]. The fact that wild Ae. aegypti are quite selective when choosing oviposition sites may be the basis for development of new strategies and products for control of dengue virus vectors. Supporting Information Table S1 Mean air temperature, relative humidity, and rainfall in Iquitos by month during 2007 through 2009. (DOC) Click here for additional data file. Checklist S1 STROBE checklist. (DOC) Click here for additional data file.

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The Nicaraguan Pediatric Dengue Cohort Study: Incidence of Inapparent and Symptomatic Dengue Virus Infections, 2004–2010

Aubree Gordon, Guillermina Kuan, Juan Carlos Mercado … (2013)

Introduction Dengue is the most common mosquito-borne viral infection worldwide, causing an estimated 40 million cases and 500,000 hospitalizations annually [1]. Most infections with the four dengue virus serotypes (DENV1-4) occur in urban and semi-urban areas of tropical and sub-tropical countries, where DENV is transmitted by Aedes aegypti and Ae. albopictus mosquitoes [2]. DENV infection results in a spectrum of disease, ranging from inapparent infection (50–90% of infections) to classic dengue fever, a self-limiting acute illness with headache, retro-orbital pain, myalgia, arthralgia, rash, and at times hemorrhagic manifestations, to life-threatening syndromes characterized by vascular leakage, hemorrhage, and shock [3]. Exposure to one serotype of DENV provides lifelong immunity to that serotype, but does not confer lasting protection to the other three serotypes; in fact, prior infection with a different DENV serotype is the single greatest risk factor for development of severe disease [4]. The earliest reports of dengue in the Americas date back to the 18th century, and dengue caused epidemics in North and South America throughout the 20th century [5]. Improved socioeconomic status, waste and water management, and behavioral factors controlled dengue in the United States, and the Pan American Ae. aegypti eradication campaign in the 1950's and '60's greatly reduced DENV transmission in Latin America [5]. However, when the program ceased, Ae. aegypti mosquitoes and DENV soon returned. In the early 1970's, only DENV-2 was endemic throughout the Americas, with limited reports of DENV-3 activity [6]. In 1977, DENV-1 was introduced, followed by DENV-4 in 1981 and an Asian DENV-3 genotype in 1994 [7]. Currently, all four serotypes circulate throughout the continent. The first reported dengue epidemic in Nicaragua occurred in 1985, when >17,000 cases and seven deaths were documented, caused by DENV-1 and DENV-2 [8]. Intensive vector control efforts resulted in no dengue outbreaks until the early 1990's, when yearly outbreaks of DENV-1, DENV-2 and DENV-4 were reported, followed by a large DENV-3 epidemic in 1994–5 [9], . Since then, all four DENV serotypes have circulated, but unlike Asia where they are hyperendemic, in Nicaragua one serotype typically dominates each season [11], [12], [13], [14], [15]. In our cohort, epidemics peak during the rainy season (especially August-January), although a low level of cases occur throughout the year [16]. DENV is the only flavivirus known to be circulating in humans in Nicaragua, and since yellow fever is not an endemic disease, the Nicaraguan population does not receive the yellow fever vaccine (A. Balmaseda, unpublished). The Pediatric Dengue Cohort Study, an ongoing prospective cohort study of dengue in children in Managua, Nicaragua, was established in August 2004 to determine the incidence of DENV infection and dengue cases, characterize the clinical spectrum of disease, and study viral and immunological determinants of DENV infection outcome [17]. A report presenting data from the first four years of the study has been published previously [16]. This paper presents results from the first six years of the Pediatric Dengue Cohort Study. Materials and Methods Ethics statement This study was conducted as a collaboration between the Nicaraguan Ministry of Health and the University of California, Berkeley. The study was approved by the Institutional Review Boards (IRBs) at the University of California, Berkeley, the Nicaraguan Ministry of Health, and the International Vaccine Initiative. Written consent was obtained from a parent or guardian, or if the guardian was illiterate, the consent form was read aloud in the presence of a witness and the guardian's thumbprint was obtained in lieu of a signature, as approved by the IRBs. Verbal assent was obtained from all children aged six years and older. Study population The Nicaraguan Pediatric Dengue Cohort Study (PDCS) is an ongoing prospective cohort study in children two to fourteen years of age in Managua, Nicaragua. A detailed description of the study design, methods, and study population has been published previously [17]. Briefly, recruitment into the study began through house-to-house visits in August 2004. All children two to nine years old living within the study area were invited to participate. At enrollment, families agreed to bring their children to the study health center, Health Center Sócrates Flores Vivas (HCSFV), for medical care at the first sign of illness. Each year, an annual blood sample was collected in July/August to enable assessment of DENV infection. The initial consent covered three years of study participation; in 2007, the study was extended for an additional three years; in 2009, the study was extended for an additional year, and in 2011, for another three years. During the first three years of the study, children were eligible to participate until they reached the age of 12. From the fourth year onwards, the protocol was modified to extend eligibility to 14 years of age. The focus of this report is the first six years of the study. The cohort was sized such that even in years of relatively low DENV transmission, a minimum number of symptomatic cases would be identified. Follow-up and case identification Participants are provided with free medical care 24 hours/day, 365 days/year through study physicians at the HCSFV. Children requiring hospitalization are transferred to the study hospital, the National Pediatric Reference Hospital (Hospital Infantil Manuel de Jesús Rivera) by study staff. At the HCSFV, children are examined by study physicians, and medical data is recorded systematically on study collection forms. Data is collected on approximately 80 variables including vital signs, temperature, musculoskeletal pain, respiratory symptoms, gastrointestinal symptoms, indicators of dehydration, and rashes and other skin anomalies. Upon medical examination, children are categorized into one of four categories according to their symptoms: suspected dengue case meeting the WHO case definition; undifferentiated febrile illness; febrile illness with defined non-dengue focus; and non-febrile illness or injury. Clinical and laboratory definitions Suspected dengue case A child presenting with a fever and two or more of the following symptoms: headache, myalgia, arthralgia, retroorbital pain, rash, hemorrhagic manifestations or leukopenia. Undifferentiated febrile illness A child presenting with an undifferentiated fever of undefined origin. Acute dengue case An ill child who tested positive for dengue as evidenced by: 1) detection of DENV RNA by RT-PCR [13], [18], 2) isolation of DENV in C6/36 cells [13], 3) seroconversion as determined by a DENV-specific immunoglobulin M (IgM) capture enzyme-linked immunosorbent assay (ELISA) using paired acute and convalescent sera [19], and/or 4) a ≥4-fold rise in total antibody titer between acute and convalescent sera as measured by Inhibition ELISA [20], [21], with titer determined using the Reed-Muench method [17], [22]. 1997 WHO case classification Dengue cases were classified according to the 1997 WHO Case Classification [3]. Dengue Fever (DF): A dengue case that does not meet the definition of DHF or DSS. Dengue Hemorrhagic Fever (DHF): A dengue case with the presence of: 1) fever or a history of fever; 2) hemorrhagic manifestations; 3) thrombocytopenia (≤100,000 platelets/ml) and 4) evidence of plasma leakage. Dengue Shock Syndrome (DSS): DHF with evidence of circulatory failure consisting of: 1) hypotension for age or narrow pulse pressure (<20 mm Hg) and 2) clinical signs of shock (e.g., rapid weak pulse, cold clammy sign, poor capillary refill) [23]. 2009 WHO case classification These criteria [24] were applied retrospectively to dengue cases that occurred in the cohort before the criteria were published. Dengue with Warning Signs: dengue cases with abdominal pain, persistent vomiting, fluid accumulation, lethargy, mucosal bleeding, fluid accumulation, liver enlargement, or increasing hematocrit with decreasing platelets. Severe Dengue.: Dengue cases with severe bleeding, severe plasma leakage leading to shock or fluid accumulation with respiratory distress, or organ failure or involvement as evidenced by liver ALT or AST ≥1,000, impaired consciousness, failure of the heart or other organs. Inapparent DENV infection A child whose paired annual serum samples demonstrated seroconversion (a titer of <1∶10 to ≥1∶10) or a ≥4-fold rise in antibody titer as determined by Inhibition ELISA and who did not experience a symptomatic DENV infection during the intervening year. Paired annual samples were run on the same ELISA plate to allow for a more accurate comparison of titers. The Inhibition ELISA has been previously evaluated in Nicaragua against the Hemagglutinin Inhibition assay [20], [21]. This evaluation showed a high concordance for seropositivity (sensitivity and specificity of the Inhibition ELISA of 98.9% and 100%, respectively) and a strong correlation of titer (Pearson's r = 0.80) between the two techniques. Moreover, to assess the reproducibility of the Inhibition ELISA assay, a subset of paired annual samples was run twice (3,510 samples corresponding to 1,755 paired annual samples). The agreement on infection outcome between the two runs was 97.9%, and the Kappa statistic was 0.836 (95% confidence interval (CI): 0.784, 0.889). Finally, the sensitivity of the Inhibition ELISA to capture DENV infections using annual samples was estimated by calculating the percentage of confirmed dengue cases that experience either seroconversion or a ≥4-fold rise in antibody titer. Of 325 dengue cases with available pre- and post-infection annual samples, 259 met the definition of a DENV infection using Inhibition ELISA; thus, the sensitivity of the assay was estimated to be 79.7% (95% CI: 74.8, 83.8). The sensitivity of the Inhibition ELISA was similar in DF cases when compared to DHF/DSS (data not shown). Primary and secondary DENV infection A DENV infection was classified as a primary DENV infection if seroconversion was observed and was considered a secondary DENV infection if a ≥4-fold increase in antibody titer was observed in paired consecutive annual samples, as determined by Inhibition ELISA [17]. If a child's serum contained anti-DENV antibody at enrollment or the child experienced a previous DENV infection during the cohort, prior to a documented subsequent DENV infection, this was also considered a secondary DENV infection. First, second, and third DENV infections were identified by counting the number of the infections documented in a participant who entered the cohort dengue-naïve, using a ≥4-fold rise in titer by Inhibition ELISA in paired annual samples to identify inapparent infections or diagnostic assays in acute and convalescent samples for symptomatic cases. The sensitivity of the Inhibition ELISA to capture primary DENV infections was estimated by calculating the seroconversion percentage in annual samples of primary dengue cases. Of 141 primary dengue cases, 125 seroconverted (sensitivity: 88.7%; 95% CI: 81.9, 93.2). Similarly, the sensitivity of the Inhibition ELISA to capture secondary DENV infections was estimated using secondary dengue cases that had demonstrated a ≥4-fold rise in titer (n = 184; sensitivity: 72.8%; 65.7, 79.0). Dengue-naïve A child who did not have detectable anti-DENV antibody at enrollment as evidenced by Inhibition ELISA and did not experience a DENV infection during the cohort study. Non-dengue-naïve A child who had anti-DENV antibody at enrollment as evidenced by Inhibition ELISA or who experienced a documented DENV infection during the cohort study. Statistical analysis Follow-up time was calculated as the amount of time between enrollment and the end of the reported study period (June 30, 2010) or withdrawal from the study. For those lost to follow-up, person-years were calculated as the time between enrollment and the last contact with study personnel, plus one-half the time between the last contact and the date recorded as lost to follow-up. Dengue cases were excluded from contributing person-time for one month following illness. Analysis of DENV infections was limited to those participants who completed the year and contributed a blood sample at the beginning and the end of the year. Analysis of second and third DENV infections was further limited to participants who entered into the cohort dengue-naïve. In order to provide conservative estimates of DENV infection incidence, since the exact timing of the DENV infection could not always be ascertained, persons who experienced a DENV infection in a given year contributed person-time for that entire year. A Poisson distribution was used to calculate 95% CIs for the incidence rates. Participant age used in the analysis of dengue case incidence was defined on a weekly basis since their exact age at the time that they were a dengue case can be ascertained. For the DENV infection analyses, participant age was defined as the age of the child when their annual sample was collected. In order to adjust for the sensitivity of the Inhibition ELISA test, each child's study ID was randomly sampled with replacement to match the observed number of children in the dataset, and then all observations from the selected children were used to create a new dataset. The expected number of DENV infections was equal to the number of observed infections in this new dataset divided by the sensitivity of the Inhibition ELISA test. The sensitivity was obtained by determining the number of symptomatic DENV infections reported in this study that were correctly identified as DENV infections by Inhibition ELISA in paired annual samples. The incidence per 1,000 person-years was 1,000 times the number of DENV infections divided by the number of person-years in the new dataset ( = 1,000*infections/(sum(days)/365.25)). This was performed 1,000 times to calculate a mean incidence and confidence intervals. Statistical analyses were performed in STATA, version 12 (StataCorp LP, College Station, TX). Results Study participation During the first six years of the PDCS (August 2004 to June 2010), 5,545 children participated, contributing 21,839 person-years. Yearly participation ranged from 3,693 to 3,953 children (Table 1), and mean participation time was 3.9 years per child (range 0.02–5.9). A total of 1,204 (21.7%) children were lost to follow-up, 341 (6.1%) children completed the time that they had consented to participate in the study and were not re-enrolled into the study, and 340 (6.1%) were withdrawn (Figure 1). Of those lost to follow-up, 844 (62.2%) had moved and could not be contacted. Annual loss to follow-up ranged from 3.8% to 6.9%. Of the 341 children who did not re-enroll in the study, 278 (81.5%) did not qualify to re-enroll either because they had reached the maximum age for participation or had moved from the study area. 10.1371/journal.pntd.0002462.g001 Figure 1 Flowchart of participants in the Pediatric Dengue Cohort Study, Managua, Nicaragua, 2004–2010. 10.1371/journal.pntd.0002462.t001 Table 1 Participant characteristics by year, Managua, Nicaragua, 2004–2010. 2004–2005 2005–2006 2006–2007 2007–2008 2008–2009 2009–2010 n = 3,721(%) n = 3,695(%) n = 3,795(%) n = 3,693(%) n = 3,953(%) n = 3,969(%) By sex Female 1,827 (49.1) 1,820 (49.3) 1,870 (49.3) 1,829 (49.5) 1,965 (49.7) 1,969 (49.6) Male 1,894 (50.9) 1,875 (50.7) 1,925 (50.7) 1,864 (50.5) 1,988 (50.3) 2,000 (50.4) By age (years) 2–5 1980 (53.2) 1635 (44.2) 1378 (36.3) 1302 (35.3) 1288 (32.6) 1103 (27.8) 6–8 1360 (36.5) 1299 (35.2) 1297 (34.2) 1231 (33.3) 1254 (31.7) 1132 (28.5) 9–14 381 (20.6) 761 (20.6) 1120 (29.5) 1160 (31.4) 1411 (35.7) 1734 (42.7) The number of participants from 2004–2005 to 2007–2008 has been previously reported [16]. Dengue cases and disease severity In Years 1–6 of the PDCS, 2,601 suspected dengue cases or undifferentiated febrile illnesses were documented, of which 351 (13.5%) were laboratory-confirmed as dengue-positive, and 138 (39.3%) children with DENV infection were hospitalized. The overall incidence rate of dengue in the cohort was 16.1 dengue cases per 1,000 person-years (95% CI: 14.5, 17.8), with yearly symptomatic incidence ranging from 3.4 to 43.5 dengue cases per 1,000 person-years. The highest incidence of dengue was observed in children ≥8 years old (Table 2, Figure 2A). A majority of cases occurred from August to February, although limited numbers of dengue cases were observed year-round (Figure 3). 10.1371/journal.pntd.0002462.g002 Figure 2 Incidence of DENV infection outcomes by age. A. Dengue cases per 1,000 person-years at risk. B. DENV infections per 1,000 person-years at risk C. Dengue cases per 100 DENV infections. 10.1371/journal.pntd.0002462.g003 Figure 3 Incidence of dengue cases in the cohort by study year and month, August, 2004–June, 2010. The number of dengue cases by month from 2004–2005 to 2007–2008 has been previously reported [16]. 10.1371/journal.pntd.0002462.t002 Table 2 Incidence of dengue cases, Managua, Nicaragua, 2004–2010. Person-years at risk Dengue cases Incidence per 1,000 person-years 95% CI All participants 21,839.3 351 16.1 14.5, 17.8 By year 2004–2005 2,942.8 17 5.8 3.6, 9.3 2005–2006 3,662.3 65 17.7 13.9, 22.6 2006–2007 3,783.7 13 3.4 2.0, 5.9 2007–2008 3,654.4 64 17.5 13.7, 22.4 2008–2009 3,887.7 22 5.7 3.7, 8.6 2009–2010 3,908.5 170 43.5 37.4, 50.6 By sex Male 11,052.0 180 16.3 14.1, 18.8 Female 10,787.3 171 15.9 13.6, 18.4 By Age (years) 2–5 7484.6 93 12.4 10.1, 15.2 6–8 7211.3 105 14.6 12.0, 17.6 9–14 7143.4 153 21.4 18.2, 25.1 The number of dengue cases from 2004–2005 to 2007–2008 has been previously reported [16]. In the first year of the study, a majority of symptomatic cases were caused by DENV-1 infection (52.9%); however, the percentage of cases caused by DENV-2 increased in 2005 through 2008 (study Years 2–4), reaching 95.3% of detected cases in 2007–2008 (Year 4) (Figure 4). DENV-3 entered the study population in 2008–2009 and was the predominant serotype in 2008–2009 (86.4%) and 2009–2010 (82.4%). Two cases of co-infection with two different DENV serotypes were also identified: a DENV-1 and DENV-4 co-infection in 2004–2005 and a DENV-1 and DENV-2 co-infection in 2005–2006 (Figure 4). A spatio-temporal video of dengue cases in the PDCS from 2004–2010 can be viewed at http://youtu.be/yQOEdsMhoSk. 10.1371/journal.pntd.0002462.g004 Figure 4 Distribution of DENV serotypes in dengue cases by study year. The percent of dengue cases infected by each DENV serotype, of the total infections with known serotype, are shown. Three hundred and thirty-one (94%) of the 351 dengue cases had serotype information available. Two cases of co-infection were detected: one DENV-1 & DENV-4 co-infection in 2004–05 and one DENV-1 & DENV-2 co-infection in 2005–06. The distribution of DENV serotypes in dengue cases from 2004–2005 to 2007–2008 has been previously reported [16]. Using the 1997 WHO definitions, of 351 dengue-positive cases, 319 (90.9%) had classic dengue fever, 21 (6.0%) had DHF, and 11 (3.1%) had DSS. The majority of these DHF/DSS cases occurred in two years of the study, with five DHF cases (23.8%) and three DSS cases (27.3%) in the 2007–2008 dengue season, and 12 DHF cases (57.1%) and seven DSS cases (63.6%) in the 2009–2010 dengue season. Using the 2009 WHO definitions of dengue severity, 182 cases were classified as dengue with warning signs (51.6% of dengue cases) and 53 (15.0%) as severe dengue. There was one death due to DENV infection during the six years of the cohort. DENV infections Analysis of DENV infections was limited to participants who completed each year and contributed a blood sample at the beginning and at the end of the year. In total, 5,073 children contributed 19,708 person-years with 1,778 DENV infections, for an incidence rate of 90.2 infections per 1,000 person-years (95% CI: 86.1, 94.5) (Table 3). The incidence of DENV infections by study year ranged from 67.0 to 119.7 infections per 1,000 person-years. Excluding 14-year-old children due to small sample size, among one-year age groups, the highest DENV infection incidence was observed in four-year old children, with an incidence of 100.4 infections per 1,000 person-years, although the incidence was fairly constant across age groups (Figure 2B). Of the 5,073 children who contributed at least one set of paired samples, 3,570 (70.4%) did not experience a DENV infection during the study, 1,250 (24.6%) experienced one documented DENV infection, 231 (4.5%) experienced two DENV infections, and 22 (0.4%) experienced three DENV infections, according to serological analysis. 10.1371/journal.pntd.0002462.t003 Table 3 Incidence of DENV infections, Managua, Nicaragua, 2004–2010. Person-years at risk DENV infections Incidence per 1,000 person-years 95% CI All participants 19,708.3 1,778 90.2 86.1, 94.5 By year 2004–2005 2,829.4 293 103.6 92.4, 116.1 2005–2006 3,426.6 410 119.7 108.6, 131.8 2006–2007 3,156.6 223 70.6 62.0, 80.6 2007–2008 3,341.9 240 71.8 63.3, 81.5 2008–2009 3,566.9 239 67.0 59.0, 76.1 2009–2010 3,386.8 373 110.1 99.5, 121.9 By sex Male 9,969.6 930 93.3 87.4, 99.5 Female 9,738.6 848 87.1 81.4, 93.1 By age 2–5 7922.1 716 90.4 84.0, 97.2 6–8 6545.9 597 91.2 84.2, 98.8 9–14 5240.2 465 88.7 81.0, 97.2 By infection number 1st 8,880.7 700 78.8 73.2, 84.9 2nd 1,137.3 138 121.3 102.7, 143.4 3rd 188.3 16 84.9 52.0, 138.7 The number of DENV infections from 2004–2005 to 2007–2008 has been previously reported [16]. The number of inapparent DENV infections in this paper vary slightly from our previous report due to the implementation of new quality control procedures which resulted in several revisions to the inapparent infection data. Over the six years, 2,779 dengue-naïve children contributed 8,881 person-years of time. The incidence of primary DENV infections was 78.8 infections per 1,000 person-years (95% CI: 73.1, 84.9) (Table 4). Yearly incidence rates ranged from 45.2 to 105.3 primary DENV infections per 1,000 person-years. Excluding 12- and 13-year-old children due to small sample size, the highest incidence rate of primary DENV infection was observed in nine-year old children (109.4; 95% CI: 81.7, 146.6). Although the highest incidence rates of primary infections were seen in older children, relatively few older children were at risk for a primary infection and therefore the majority of DENV infections in older children were secondary (Figure 5). 10.1371/journal.pntd.0002462.g005 Figure 5 Percentage of primary and secondary DENV infections in the cohort by year of age. The percentage of primary and secondary immune responses, by age, from 2004–2005 to 2007–2008 has been previously reported [16]. 10.1371/journal.pntd.0002462.t004 Table 4 Incidence of primary and secondary DENV infections, Managua, Nicaragua, 2004–2010. Primary DENV Infections Secondary DENV Infections Person-years at risk DENV infections Incidence per 1,000 person-years 95% CI Person-years at risk DENV infections Incidence per 1,000 person-years 95% CI All participants 8,880.7 700 78.8 73.2, 84.9 10,829.6 1,078 99.5 93.8, 105.7 By year 2004–2005 1,162.1 98 84.3 69.2, 102.8 1,667.3 195 117.0 101.6, 134.6 2005–2006 1,408.3 148 105.1 89.5, 123.5 2,018.3 262 129.8 115.0, 146.5 2006–2007 1,382.6 80 57.9 46.5, 72.0 1,774.0 143 80.6 68.4, 95.0 2007–2008 1,547.7 123 79.5 66.6, 94.8 1,794.2 117 65.2 54.4, 78.2 2008–2009 1,746.6 79 45.2 36.3, 56.4 1,821.4 157 87.8 75.2, 102.6 2009–2010 1,633.4 172 105.3 90.7, 122.3 1,754.4 180 114.5 99.8, 131.6 By sex Male 4,460.7 352 78.9 71.1, 87.6 5,510.9 578 94.0 86.1, 102.6 Female 4,419.6 348 78.7 70.9, 87.4 5,318.7 500 104.9 96.7, 113.8 By age 2–5 5673.7 427 75.3 68.4, 82.7 2248.5 288 128.1 114.1, 143.8 6–8 2409.2 190 78.9 68.4, 90.9 4138.6 408 98.6 89.5, 108.6 9–14 797.9 83 104.0 83.9, 129.0 4442.4 382 86.0 77.8, 95.1 The number of primary and secondary DENV infections from 2004–2005 to 2007–2008 has been previously reported [16]. The 2,813 non-dengue-naïve children contributed 10,830 person-years. The incidence of secondary DENV infections was 99.5 infections per 1,000 person-years (95% CI: 93.8, 105.7) (Table 4), with annual secondary infection incidence rates ranging from 59.6 to 115.2 infections per 1,000 person-years. The highest incidence of secondary infections was observed in children aged five and under. However, when the percentage of primary and secondary infections was examined by year of age (Figure 5), the majority of DENV infections in younger children were primary, presumably due to the relatively small percentage of young children at risk for a secondary infection. To examine the potential effects of both waning antibody levels and the use of the Inhibition ELISA assay on our estimate of overall DENV infection incidence, we performed a sensitivity analysis by determining how many symptomatic DENV infections reported in this study were correctly identified as DENV infections by Inhibition ELISA in paired annual samples. This yielded a sensitivity of 79.7%, which we then applied to our observed estimate of incidence to arrive at an incidence rate of 112.5 DENV infections per 1,000 person-years (95% CI: 107.7, 117.4). Therefore, our observed incidence is a conservative estimate of the true incidence in the cohort. Repeat DENV infections Of the 700 children who entered the cohort dengue-naïve and experienced a primary DENV infection, 138 went on to experience a second DENV infection. The 700 children contributed 1,137 person-years of time, yielding an incidence rate of 121.3 second DENV infections per 1,000 person-years (95% CI: 102.7, 143.4) (Table 3). Of the 138 children who contributed 188.3 person-years of time at risk, 16 experienced third DENV infections, for an incidence rate of 84.9 third DENV infections per 1,000 person-years (95% CI: 52.0, 138.7) (Table 3). There were no fourth DENV infections observed during the 8.0 person-years of time at risk for a fourth infection. Dengue cases among DENV infections The overall rate of symptomatic cases among DENV infections was 18.2 dengue cases per 100 DENV infections (Table 5). This rate varied dramatically by year; from 4.9 cases per 100 infections (95% CI: 2.7, 8.9) in 2006–2007 to 40.8 cases per 100 infections (95% CI: 34.8, 47.8) in the 2009–2010 season. The lowest rates of symptomatic cases were seen in the youngest age groups, two- and three-year olds, with rates of 11.8 and 10.8 cases per 100 infections, respectively. The highest rates were observed in the older age groups, especially in children aged nine and over, with rates of 23.6 to 29.9. 10.1371/journal.pntd.0002462.t005 Table 5 Incidence of dengue cases among DENV infections, Managua, Nicaragua, 2004–2010. DENV infections Dengue cases Incidence of cases per 100 DENV infections 95% CI All participants 1,778 325 18.2 16.4, 20.4 By year 2004–2005 293 17 5.8 3.6, 9.3 2005–2006 410 64 15.6 12.2, 19.9 2006–2007 223 11 4.9 2.7, 8.9 2007–2008 240 60 25.0 19.4, 32.2 2008–2009 239 21 8.8 5.7, 13.5 2009–2010 373 152 40.8 34.8, 47.8 By sex Male 930 166 17.8 16.1, 21.9 Female 848 159 18.9 15.3, 20.7 By age (years) 2–5 716 98 13.7 11.2, 16.7 6–8 597 100 16.8 13.8, 20.4 9–14 465 127 27.3 23.0, 32.4 Discussion To date, this is one of the longest continuous, large-scale, community-based prospective cohort study to characterize dengue cases and DENV infections, and it is currently ongoing. We show that there is a high incidence of DENV transmission in children in Managua, Nicaragua, leading to a substantial incidence of dengue cases, a large proportion of which were hospitalized. Although quite variable year-to-year, on average approximately six times as many DENV infections as cases were documented, illustrating that the number of symptomatic cases substantially underestimates DENV transmission in Nicaragua, consistent with our previous report on this cohort [16] and reports from other regions [16], [25], [26], [27]. Several prospective cohort studies of dengue and DENV infection in Asian and Latin American countries have been reported [16], [25], [27], [28], [29], [30], [31], [32], [33]. In Indonesia, during a one-year period of a study in children 4–9 years old, 23.2% of children were reported to have experienced a DENV infection [28]. In a seven-month dengue season during a prospective serological study in Thai schoolchildren aged 4–16 years, 5.6% of the children experienced a DENV infection, of whom 86% presented as asymptomatic or minimally symptomatic (defined as absent from school for one day) [29]. In a three-year prospective cohort study of DENV infections in Thai elementary schoolchildren, 5.8% of the children were infected with DENV, with 53% of infections presenting as inapparent DENV infections [25]. In a four-year prospective study of DENV infections in Thai children, 2.3% of children experienced a DENV infection per season, and the ratio of inapparent-to-symptomatic infection was 1.8∶1 [34]. In Iquitos, Peru, an incidence rate of 20–30 DENV-1 or DENV-2 infections per 1,000 person years was observed in a 3.5 year-long cohort study of ∼2,400 children and adults in years where the population had previously been exposed to DENV-1 or DENV-2; however, a peak rate of 890 infections per 1,000 person-years was observed with the introduction of DENV-3 into this population that was completely susceptible to DENV-3 [32]. The overall incidence of DENV infections (90.2 per 1,000 person-years) observed in this study in Nicaragua was within the range of the estimates reported in studies in Asia, while the incidence of dengue cases (16.1 per 1,000 person-years) was in general lower than observed in Asia. The incidence rate of second DENV infections in the Nicaraguan PDCS was significantly higher than that of primary infections, presumably due to the fact that children who have already experienced a primary DENV infection and are at risk for second DENV infections live in areas where they are more likely to be exposed to DENV than the overall dengue-naïve study population, who are at risk for primary DENV infections. The rates of second and third DENV infections were not significantly different. In this manuscript, two dengue classification schemes, from the 1997 and the 2009 WHO Guidelines, were used [3], [24]. This allows for comparison to historical as well as future studies. The percentage of dengue cases qualifying as severe dengue (15%) or dengue with signs of alarm (53%) was higher than those meeting the traditional DHF/DSS classification (9%), consistent with other reports [35]. Overall, the observed rate of dengue cases among DENV infections was 18.2 cases per 100 infections (range 4.8–40.8 cases per 100 DENV infections), and a clear pattern of alternating seasons of high and low dengue case incidence was observed. This pattern was not due to differences in DENV infection rates, but rather to differences in the rate of dengue cases per DENV infection. As reported in the first four years of this study [16] and contrary to what has been described in Thailand [25], [26], [27], no positive correlation between the incidence of DENV infections and the incidence of dengue cases among DENV infections by year was observed. There were two years in particular where the rate of dengue cases per 100 DENV infections was high, the 2007–2008 dengue season (25.0 infections per 100 person-years) and the 2009–2010 dengue season (40.8 cases per 100 DENV infections). An increase in disease severity was also observed in the same two years [15], [36]. In 2007–2008, several factors appear to have contributed: waning DENV-1 immunity followed by DENV-2 resulted in more disease severity after a period of approximately two years, plus a clade replacement in DENV-2 led to greater disease severity in DENV-3-immune children infected by the replacing DENV-2 clade, NI-2B [15]. In 2009–2010, an unusual overlap with influenza pandemic strain H1N1 may have led to the observed atypical dengue presentation, more symptomatic DENV infections, and greater disease severity [36]. In this study, the highest incidence of primary infections was observed in older dengue-naïve children. This seems counter-intuitive, as the majority of DENV infections in older children are secondary (Figure 5). However, one possible reason for the higher rates of primary infections in older dengue-naïve children is that older children spend more time away from the house and thus have more opportunity to encounter DENV-infected mosquitoes both in the house and in other venues. Interestingly, although the majority of secondary DENV infections are in older children, the highest incidence rates of secondary infections were observed in the youngest children. Two possible explanations for this are first, that older non-dengue-naïve children may have already experienced more than one previous DENV infection and therefore have increased immunity (type-specific immunity to more serotypes and presumably some level of cross-reactive immunity), whereas younger non-dengue-naïve children are more likely to have only experienced a single prior DENV infection and are therefore more likely to be susceptible to the circulating DENV serotype. In fact, some of the older children may have experienced infection with all four DENV serotypes and therefore may not be at risk for infection. However, since many children enter the cohort non-naïve, we are unable to determine who is no longer at risk and therefore should not contribute person-time. Another possible explanation is that there may be a small percentage of the general population that is at greatest risk for DENV infection due to environmental or behavioral risk factors and therefore some children may experience multiple infections at an early age, thereby increasing the rate of secondary infection in some younger children. These findings highlight the importance of analyzing infection incidence rather than only proportions. The strengths of this study include its large size, low loss to follow-up, high participation rates, and year-round follow-up. Many dengue cohort studies only operate during the dengue season, which results in an underestimate of annual incidence of dengue. The rigorous analysis used here, including incidence reporting in person-years, allows comparison across studies and across different infectious diseases. We are currently investigating factors that affect the incidence of symptomatic cases among DENV infections. Another strength of the study is the extensive use of information technologies and intensive quality control procedures since the study's inception [17]. Limitations of the study include ascertainment of cases through enhanced passive surveillance. Thus, some dengue cases may not have been detected due to parents not bringing their child in for healthcare [17]. Another limitation is that serum samples for cohort-wide serological testing were only available from participants once per year; however, due to the longevity of the study, more frequent sampling is not tolerated well by cohort participants. In this study, a ≥4-fold rise in antibody titer in paired annual samples was used to define a DENV infection. It is known that antibody levels begin to wane several months post-infection in dengue cases, but little to no information is available about antibody kinetics in inapparent DENV infections. A third limitation is that our annual sample was collected in July/August of each year, and during several of the study years, dengue transmission began during the sampling period. Thus, it is possible that some individuals may have been sampled very close to a DENV infection. This would result in misclassification of their infection by study year or classification as a DENV infection both in both years (in those individuals whose antibody levels were still rising at the time of sampling) when in fact they only experienced one DENV infection. Additionally, we exclude acute dengue cases for one month following infection, which makes incidence estimates conservative as children are likely protected from re-infection for substantially longer. This study relies on Inhibition ELISA to assess inapparent infections. Although no gold standard formally exists for assessing inapparent DENV infection, the plaque reduction neutralization test (PRNT) is considered the best method. However, PRNT is extremely labor-intensive, and given the resources available for the study, PRNT or other flow-based neutralization assays [37], [38] were not a feasible laboratory technique for a cohort of this size. Finally, as this study is limited to children, we are unable to provide information on the burden of dengue in adults in Nicaragua; however, establishing the incidence of dengue in children is particularly important as it is expected that they will be the first target population once there is a licensed dengue vaccine. In summary, we have reported substantial incidence of DENV infections and dengue cases in children in Nicaragua using rigorous analytic methods. We have included several additional years of the cohort study and have presented the data in a form that can be readily used for comparison with incidence rates for other studies or other diseases and extrapolation for burden of disease estimates. Although costly and labor-intensive, cohort studies are the only way to determine the incidence of DENV infection and one of the few ways to capture the true rate of disease, which is greatly underestimated via national passive surveillance systems [39], [40]. These data are then utilized as a benchmark for establishing expansion factors that are used to estimate the actual disease burden [39], [41], [42] and the economic cost of the disease [43], [44], [45], as well as for cost-effectiveness studies of vaccines or other interventions [46], [47]. Thus, our incidence estimates have significant policy implications for dengue vaccines as they become available. Supporting Information Checklist S1 STROBE Checklist for cohort studies. (DOCX) Click here for additional data file.

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Jorge Arosteguí: ciet@cablenet.com.ni

Josefina Coloma: colomaj@berkeley.edu

Carlos Hernández-Alvarez: carher@ibw.com.ni

Harold Suazo-Laguna: hsuazo_laguna@hotmail.com

Angel Balmaseda: abalmaseda@minsa.gob.ni

Eva Harris: eharris@berkeley.edu

Neil Andersson: andersson@ciet.org

Robert J Ledogar: rledogar@ciet.org

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Journal ID (iso-abbrev): BMC Public Health

Title: BMC Public Health

Publisher: BioMed Central (London )

ISSN (Electronic): 1471-2458

Publication date (Electronic): 30 May 2017

Publication date PMC-release: 30 May 2017

Publication date Collection: 2017

Volume: 17

Issue : Suppl 1 Issue sponsor : Publication of this supplement has been funded by the UBS Optimus Foundation. The articles have undergone the journal's standard peer review process for supplements. The Supplement Editor declares no competing interests.

Electronic Location Identifier: 434

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[1 ]CIET International, Managua, Nicaragua

[2 ]ISNI 0000 0001 2181 7878, GRID grid.47840.3f, Division of Infectious Diseases and Vaccinology, School of Public Health, , University of California, ; Berkeley, CA USA

[3 ]GRID grid.419860.2, Centro Nacional de Diagnóstico y Referencia, , Ministerio de Salud, ; Managua, Nicaragua

[4 ]ISNI 0000 0001 0699 2934, GRID grid.412856.c, Centro de Investigación de Enfermedades Tropicales, , Universidad Autónoma de Guerrero, ; Acapulco, Guerrero Mexico

[5 ]ISNI 0000 0004 1936 8649, GRID grid.14709.3b, Department of Family Medicine, , McGill University, ; Montreal, Canada

[6 ]CIET International, New York, NY USA

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DOI: 10.1186/s12889-017-4296-6

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PubMed ID: 28699558

SO-VID: 783209b4-0f01-4878-9e66-bea756ac27a5

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Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Conference name: The Camino Verde Trial colloquium

Conference location: Acapulco, Mexico

Conference date: 17-21 June 2013

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Keywords: temephos,clusters,aedes aegypti,camino verde,dengue prevention

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Keywords: temephos, clusters, aedes aegypti, camino verde, dengue prevention

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Beyond efficacy in water containers: Temephos and household entomological indices in six studies between 2005 and 2013 in Managua, Nicaragua

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