Serotype-Specific Epidemiological Patterns of Inapparent versus Symptomatic Primary Dengue Virus Infections: A 17-year cohort study in Nicaragua
Authors: Bos Sandra , Zambrana Jose Victor , Duarte Elias , Graber Aaron L. , Huffaker Julia , Montenegro Carlos , Premkumar Lakshmanane , Gordon Aubree , Kuan Guillermina , Balmaseda Angel , Harris Eva 
Published: April 2024

AbstractBackgroundDengue is the most prevalent mosquito-borne viral disease and a major public health problem worldwide. Most primary infections with the four dengue virus serotypes (DENV1-4) are inapparent; nonetheless, whether the distribution of symptomatic versus inapparent infections by serotype varies remains unknown. Here, we present (1) the evaluation of a multiplex DENV1-4 envelope domain III multiplex microsphere-based assay (EDIII-MMBA) to serotype inapparent primary infections and (2) its application leveraging 17 years of prospective sample collection from the Nicaraguan Pediatric Dengue Cohort Study (PDCS).MethodsFirst, we evaluated the performance of the EDIII-MMBA with samples characterized by RT-PCR or focus reduction neutralization test. Next, we analyzed 46% (N=574) of total inapparent primary DENV infections in the PDCS with the EDIII-MMBA to evaluate the epidemiology of inapparent infections. Remaining infections were inferred using stochastic imputation, taking year and neighborhood into account. Infection incidence and percentage of inapparent, symptomatic, and severe infections were analyzed by serotype.FindingsThe EDIII-MMBA demonstrated excellent overall accuracy (100%, 95·8-100%) for serotyping symptomatic and inapparent primary DENV infections when evaluated against gold-standard serotyping methods. We found that a significant majority of primary infections were inapparent, with DENV3 exhibiting the highest likelihood of symptomatic and severe primary infections (Pooled OR compared to DENV1 = 2·13, 95% CI 1·28-3·56, and 6·75, 2·01-22·62, respectively), whereas DENV2 was similar to DENV1 in both analyses. Significant within- and between-year variation in serotype distribution between symptomatic and inapparent infections and circulation of serotypes undetected in symptomatic cases were observed in multiple years.InterpretationOur study indicates that case surveillance skews the perceived epidemiological footprint of DENV. We reveal a more complex and intricate pattern of serotype distribution in inapparent infections. The significant differences in infection outcomes by serotype emphasizes the need for vaccines with balanced immunogenicity and efficacy across serotypes.FundingNIH/NIAID P01AI106695, U01AI153416Research in contextEvidence before this studyWe conducted a search in PubMed for studies published up to February 2024. Keywords included “dengue virus” and “DENV” in combination with “inapparent infections”, “asymptomatic infections”, “primary infections by serotype”, “FoI by serotype”, “force of infection”, “force of infection by serotype”, and identified a significant gap in the current understanding of dengue epidemiology. Despite acknowledging the high prevalence of inapparent DENV infections in endemic regions, previous research has focused primarily on symptomatic infections, potentially biasing our understanding of the DENV epidemiological landscape and hindering our capacity to determine the complete disease spectrum of the different DENV serotypes. While cross-sectional studies have provided preliminary insights into this gap, there is a need for more comprehensive and detailed serotype-specific insights to evaluate the epidemiological impact of inapparent infections. The lack of comprehensive characterization of inapparent infections reflects methodological challenges, particularly the need for prospective cohort studies designed to capture and accurately serotype these infections. Moreover, the reliance on labor-intensive and low-throughput antibody neutralization assays for serotyping, despite their accuracy, has constrained high-throughput analysis required for large-scale epidemiological studies.Added value of this studyOur study, spanning 17 years of prospective cohort data in Nicaragua, addresses this bottleneck in dengue research by providing a detailed examination of primary inapparent infections. The introduction of a novel envelope domain III (EDIII) multiplex microsphere-based assay for DENV serotyping represents a significant methodological advance, offering an efficient, scalable alternative for large epidemiological studies. A key contribution of our study is the intricate pattern of serotype distribution among inapparent infections. In contrast to the serotype predominance observed in symptomatic infections, inapparent infections exhibit a complex landscape with co-circulation of multiple DENV serotypes, including serotypes undetected in symptomatic surveillance in multiple years. Our systematic documentation of the entire disease spectrum provides unprecedented insights into the serotype-specific disease burden in primary infection, including the proportion of symptomatic versus inapparent infection and its temporal variations, thus providing a more complete picture of DENV epidemiology than has been available to date. Notably, we demonstrate striking differences in disease severity by serotype, with DENV3 infections being significantly more symptomatic and more severe compared to DENV1 and DENV2, the latter displaying the highest rate of inapparent infection.Implications of all the available evidenceOur research challenges prior assumptions by demonstrating that inapparent and symptomatic primary DENV infections present distinct epidemiological profiles, revealing that the epidemiological footprint of DENV is broader and more nuanced than previously recognized through symptomatic cases alone. These findings underscore the utility for continuous and comprehensive surveillance systems that capture both symptomatic and inapparent infections to accurately assess the epidemiological burden of DENV and inform public health interventions. Additionally, they provide critical insight for enhancing the accuracy of predictive DENV transmission modeling. Furthermore, the marked differences in infection outcomes by serotype emphasize the need for serotype-informed public health strategies. This nuanced understanding is pivotal for the crafting of targeted interventions, vaccine development and vaccination strategies, and efficient resource allocation, ultimately contributing to the global effort to mitigate the impact of dengue.