Serotype-Specific Epidemiological Patterns of Inapparent versus Symptomatic Primary Dengue Virus Infections: A 17-year cohort study in Nicaragua

Abstract

BackgroundDengue 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; nevertheless, prior research has primarily focused on symptomatic infections, which has limited our understanding of the epidemiological burden and spectrum of disease of each DENV serotype. Our study addresses this bottleneck in dengue research by providing a new method and a detailed examination of primary inapparent infections.MethodsHere 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. After evaluation, we analyzed 46% (N=574) of total inapparent primary DENV infections with the EDIII-MMBA. Remaining infections were inferred using stochastic imputation, taking year and neighborhood of infection into account.FindingsThe EDIII-MMBA demonstrated excellent diagnostic accuracy of symptomatic and inapparent primary DENV infections when evaluated against gold-standard serotyping methods. 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. Our findings reveal that a significant majority of primary infections remained inapparent: 76.8% for DENV1, 79.9% for DENV2, and 63.9% for DENV3. DENV3 exhibited 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 had similar likelihood to DENV1 in both analyses.InterpretationOur study indicates that case surveillance skews the perceived epidemiological footprint of DENV and reveals a more complex and intricate pattern of serotype distribution in inapparent infections. Further, the significant differences in infection outcomes by serotype emphasizes the need for serotype-informed public health strategies.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.

Complement-dependent virion lysis mediated by dengue-Zika virus cross-reactive antibodies correlates with protection from severe dengue disease

“…In the present study, which includes presecondary DENV1, DENV2, and DENV3 infection samples, after performing a stratification analysis by DENV serotype, we found that ADCD was correlated with protection especially in the pre-secondary DENV3 infection sample group, using E and NS1 recombinant antigens. In contrast, we found that nAb titers are more correlated with protection in pre-DENV2 compared to pre-DENV3 infection samples (36), raising the possibility that protection against different DENV serotypes may be associated with different antibody functions. When the ADCD assay was performed with ZIKV E and NS1 antigens, protection was observed when all samples were analyzed together, but not when the analysis was stratified by pre-DENV2 and pre-DENV3 subgroups.…”

“…Currently, neutralization assays have been the main method used to assess protection in vaccine trials and natural infection studies, with the caveat that nAb titers do not always correlate with protection from disease in humans. Multiple parameters (e.g., virion maturation state, cell line substrate, infection history, and incoming infecting serotypes) have been shown to affect neutralization assays, highlighting the need to control assay conditions carefully and to develop novel methods to assess protection ( 22 , 36 ). The complex nature of anti-DENV antibodies and the lack of tools to indicate individuals protected from disease have led to major shortcomings in vaccine development.…”

“…DF controls were randomly matched with severe cases by serotype and infection history, applying frequency matching. Cases and controls were restricted to individuals without prior exposure to the incoming serotype or ZIKV as confirmed by various methods such as reverse transcriptase-polymerase chain reaction (RT-PCR), nAb titers, epidemiological data, inhibition enzyme-linked immunosorbent assay (iELISA) binding antibody titers, ZIKV BOB ELISA, and binding to envelope (E) protein domain III (EDIII) in a Luminex assay ( 5 , 14 , 36 , 48 ). Samples obtained after a single DENV infection are referred to as post-primary infection samples, while samples obtained after two or more prior DENV infections are referred to as post-secondary infection samples ( Fig.1 ).…”