Development of Inapparent Dengue Associated With Increased Antibody Levels to Aedes aegypti Salivary Proteins: A Longitudinal Dengue Cohort in Cambodia

Abstract

Background: We established the first prospective cohort to understand how infection with dengue virus is influenced by vector-specific determinants such as humoral immunity to Aedes aegypti salivary proteins.

Methods: Children aged 2-9 years were enrolled in the PAGODAS (Pediatric Assessment Group of Dengue and Aedes Saliva) cohort with informed consent by their guardians. Children were followed semi-annually for antibodies to dengue and to proteins in Ae. aegypti salivary gland homogenate using enzyme-linked immunosorbent assays and dengue-specific neutralization titers. Children presented with fever at any time for dengue testing.

Results: From 13 July to 30 August 2018, we enrolled 771 children. At baseline, 22% (173/770) had evidence of neutralizing antibodies to 1 or more dengue serotypes. By April 2020, 51 children had symptomatic dengue while 148 dengue-naive children had inapparent dengue defined by neutralization assays. In a multivariate model, individuals with higher antibodies to Ae. aegypti salivary proteins were 1.5 times more likely to have dengue infection (hazard ratio [HR], 1.47 [95% confidence interval {CI}, 1.05-2.06]; P = .02), particularly individuals with inapparent dengue (HR, 1.64 [95% CI, 1.12-2.41]; P = .01).

Conclusions: High levels of seropositivity to Ae. aegypti salivary proteins are associated with future development of dengue infection, primarily inapparent, in dengue-naive Cambodian children.

Clinical trials registration: NCT03534245.

Keywords: Aedes aegypti; Cambodia; dengue; mosquito saliva; pediatric cohort.

Conflict of interest statement

Potential conflicts of interest. All authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.

Published by Oxford University Press for the Infectious Diseases Society of America 2021.

Figures

Figure 1.

Study flowchart. Seven hundred seventy-five children were screened for enrollment into the cohort; 771 children were enrolled and followed up every 6 months (± 1 month) for pan–dengue virus (DENV) and Aedes aegypti salivary gland homogenate enzyme-linked immunosorbent assay (ELISA) screening for seroprevalence. In between visits, children had semi-active surveillance for dengue by being encouraged to visit the hospital for febrile episodes. Children with symptomatic polymerase chain reaction–confirmed dengue were hospitalized per Cambodian national guidelines. Clinically inapparent dengue infections were any children who seroconverted via pan-DENV ELISA screen then confirmed via DENV1–4 50% plaque reduction neutralization test titers >1:40, but never had a clinically apparent or symptomatic DENV episode. Abbreviations: DENV, dengue virus; IgM, immunoglobulin M; PCR, polymerase chain reaction.

Figure 2.

Seroprevalence to dengue and Aedes aegypti salivary proteins. Seroprevalence to dengue and Aedes aegypti saliva over the first 24 months of study follow-up with sampling every 6 months alternating rainy and dry seasons (eg, baseline and visit 3 are rainy seasons). A, Serotype-specific dengue seroprevalence confirmed by neutralization assays, by age. B, Binary antibody response to Aedes aegypti salivary proteins (higher/lower). Abbreviations: DENV, dengue virus; SGH, salivary gland homogenate.

Figure 3.

Hazard risk of Aedes aegypti salivary protein antibody levels as predictor of dengue infection in a dengue-naive population at baseline. Cox proportional hazards model showing the effects of higher (green) vs lower (blue) Ae. aegypti mosquito salivary protein antibody levels on the risk of dengue infection (A); and the effect of Ae. aegypti salivary protein antibody levels on the risk of inapparent dengue (green and blue) vs apparent, or symptomatic, dengue (red and orange) (B). Steep increases every 6 months are artifact of sampling scheme for inapparent dengue cases compared to apparent cases that were diagnosed on a rolling basis. Abbreviations: Ab, antibody; DENV, dengue virus.

Figure 4.

Map of study area and visual description of geospatial model of antibody levels to Aedes aegpyti salivary proteins on predicting dengue risk. A, Aerial view of study site (designated by green lines) with hospital (red cross). B, Smoothed surface depicting intensity of participants’ seroreactivity to Ae. aegypti salivary proteins, generated from optical density values. C, Grid cells (100 m × 100 m) and mean Ae. aegypti salivary protein antibody values at the grid cell level (higher values are darker red), with both apparent and inapparent dengue virus infections overlaid (black crosses). Abbreviation: DENV, dengue virus.