Dengue virus (DENV), transmitted by female Aedes mosquitoes, represents a challenging global public health concern (1, 2). Globally, an estimated 3.9 billion people have become infected, mainly in the tropical and sub-tropical regions, with an eightfold increase in incidence reported in the last two decades (3). Notwithstanding that DENV infections are often asymptomatic, exacerbation could result in severe dengue (4). Four antigenically distinct serotypes, i.e., DENV1–DENV4, circulate in the population, while a fifth sylvatic serotype reportedly circulate among non-human primates (5, 6).

The disease spectrum and severity are protean and may vary from unapparent infection to life-threatening severe dengue, often marked by severe thrombocytopenia and multi-organ failure (7). Progression of the disease from mild to severe dengue is influenced by various factors like heterologous virus serotype and host inflammatory responses (8, 9). The 1997 WHO classification stratified dengue into dengue fever (DF), dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS), with a DF diagnosis emphasizing the requirement of laboratory confirmation. Furthermore, as per the revised 2009 WHO classification, dengue infection is also categorized (based on disease severity) as dengue with warning signs (DWS+), dengue without warning signs (DWS-), and severe dengue. DWS+ is characterized by symptoms/signs, including abdominal pain, recurrent vomiting, lethargy, hepatomegaly, hemoconcentration, and thrombocytopenia, whereas severe dengue includes severe plasma leakage, hemorrhage, and organ failure (10).

Of the various inflammatory pathways, the complement activation cascade holds key in dengue pathogenesis, and its role in clinical dengue largely remains obscure (11). The complement system bridges the innate and the adaptive immune systems (12, 13) via three major pathways, namely, the classical, the alternative, and the lectin pathways (14), and also the blood coagulation pathway. Complement activation culminates in heightened plasma levels of anaphylatoxins (especially C3a, C4a, and C5a), driving the infiltration of polymorphonuclear neutrophils (PMNs) and natural killer (NK) cells, leading to excessive release of pro-inflammatory cytokines, which, at times, could entail endothelial dysfunction (i.e., vasodilation and increased vascular permeability) (15).

The complement system appears to play a role in dengue pathogenesis by inducing inflammation and tissue/organ damage. The mannose-binding lectin (MBL) pathway appears to limit viral replication in asymptomatic (self-limiting) and primary dengue (16). Elevated complement anaphylatoxins have been reported in severe secondary dengue, whereas soluble immune complexes during acute dengue disease will become bound by CR1 (CD35) expressed on erythrocytes (17) for disposal in the spleen. Owing to its regulatory role, CR1 (also designated as CD35) appears to limit inflammation and tissue damage (18). Despite these insights, there remains a lack of direct clinical data correlating complement levels with dengue disease severity. Here we quantitatively assessed key complement proteins and a myriad of other clinical laboratory parameters in dengue. We determined if complement factors and acute-phase reactants correlated with disease severity attributing to improved understanding of dengue pathogenesis and endeavored to identify surrogate biomarkers/therapeutic targets in severe dengue.

In the current cross-sectional case–control investigation, we surveyed the role of various clinical laboratory parameters and immunological determinants, with particular emphasis on the complement system in DENV across varying grades of disease severity. Of the 156 study participants, 114 cases were serologically confirmed cases of DENV infection, with a predominance of IgM positivity and a small number of NS1-positive and secondary dengue cases. Viral RNA was also present in a minority of samples, all of which were NS1-positive, reinforcing the utility of NS1 as a marker of acute dengue viremia (22, 23). The low percentage of samples with dengue viremia could be attributed to the temporospatial inaccuracies in specimen collection (likely during the late acute phase (between days 3 and 5 of the onset of fever) when viremia has typically reduced, rather than assay failure. This is consistent with established dengue kinetics, where viral RNA falls rapidly in 4 to 5 days of illness (24).

Analysis of complement components across stages of infection revealed distinct alterations in the early classical complement proteins. C1Inh (regulatory), C1q, and C2 demonstrated severity-dependent variations, suggesting that the classical pathway plays a crucial role in dengue immunopathogenesis. Our observations are consistent with prior evidence indicating that the formation of an immune complex and antibody-mediated response can trigger the activation of a classical pathway in dengue (25, 26). Antibody-dependent enhancement (ADE) of dengue virus infection has been one of the main hypotheses to explain the disease severity of dengue (26). In the presence of sub-neutralizing antibody, immune complexes can amplify the viral replication in phagocytic cells, which will, in turn, activate the complement system via cytokine cascade (9, 27). The elevated levels of C1Inh likely represent a host’s compensatory response aimed at counteracting excessive complement activation, thereby limiting the inflammation and the likely vascular activation mechanisms triggered (28). Similarly, the attrition in C1q levels is consistent with its consumption during immune-complex-driven activation, a well-defined hallmark of dengue disease, especially during secondary infection (26, 29).

Our study also observed that MBL did not have any significant differential expression across primary and secondary dengue groups, although it did show a differential expression between varying grades of dengue disease severity. This observation, in turn, is consistent with the functional biology of MBL as a lectin complement pathway protein that recognizes carbohydrate moieties (23, 30). Thus, the expression of MBL is not expected to vary remarkably between primary and secondary dengue, which primarily differentiates between humoral immune responses. On the contrary, the association of MBL with disease severity highlights its role in modulating host–virus interactions and complement activation during acute infection, making it more marker-associated with clinical outcome and not of disease severity (17, 30). Our investigations also showed that the downstream complement activation products, i.e., C3a, C3b, and C5a, and the complement regulatory protein sCR1 did not show any significant difference in expression in varying grades of disease severity, i.e., DWS+, DWS-, and severe dengue as well as primary/secondary dengue. This suggests that terminal complement activation is not uniformly altered during disease severity, possibly due to efficient regulation or localized activation.

The selective alterations in C1q, C1Inh, and C2 levels in overall patient cohort point to a preferential engagement of the classical pathway during early DENV infection. Significant shifts in the proximal classical complement factors and the lack of marked alterations in downstream components suggest the likely initiation of the classical pathway but not fully propagated across to the terminal effectors of complement activation (involving C6, C7, C8, and C9, which, however, could not be completely explored in the current study). This could still be speculated of the likely involvement of early immune-complex-driven activation but are tightly regulated, which remains to be explored. In contrast, a minimal variation in MBL in primary/secondary dengue cohort while indicating significant alterations in the dengue cohort indicates that the lectin pathway may seldom contribute to complement activation. This supports a suggestive interplay, where classical pathway may likely serve as the main axle of complement activation in DENV infection (17, 31).

One of the most important observations was the association between sCR1 and disease severity. By univariate analysis, several parameters, including age, NS1 positivity, and IgG positivity, were found to be associated with dengue severity, while multivariate modeling identified sCR1 as the only factor associated with dengue disease severity. Furthermore, higher sCR1 levels were protective, with each unit increase of sCR1 reducing the dengue severity by 22%. This suggests that sCR1 exhibits a regulatory role in controlling complement activation and preventing excessive inflammation and tissue injury during systemic infection (26, 32). Many previous studies have implicated uncontrollable complement activation as a contributor to plasma leakage, and our study hypothesizes the role of sCR1 as a protective regulator (33). In addition, sCR1 showed a negative association with platelet count but which did not reach statistical significance, implicating that its suggestive role is more closely related to dengue severity than to thrombocytopenia. It is important to note that the complement system also links to the coagulation systems, and hence the association between sCR1 and platelet requires greater attention. CR1 is a type I membrane protein that encompasses a family of complement receptors (CR2, CR3, and CR4) that are often expressed on a plethora of immune cells (34). Besides that, very low levels (~50 ng/mL) of CR1 occur as sCR1 in the plasma, likely released as a result of proteolytic shedding (34).

Hematologic and biochemical results associated with the platelet count also provided additional insights. In line with previous findings (35, 36), markers of immune dysregulation, such as RDW and basophil levels, were inversely correlated with the platelet levels. These findings are consistent with the ongoing hematologic derangement occurring in dengue thrombocytopenia (35, 36). Interestingly, uric acid levels showed a strong positive correlation with platelet count, raising the probability that altered purine metabolism or oxidative stress pathway can influence the hematologic recovery in DENV infection (37). However, this observation is only preliminary and requires further in-depth investigations. Uric acid functions as a major endogenous antioxidant, scavenging ROS and protecting the host against cellular damage (38). The observed positive correlation between uric acid and platelet levels and its implied negative correlation between dengue severity reflect excessive oxidative stress and antioxidant depletion, implying an uptake of uric acid metabolites and a decreased capacity of endogenous antioxidant defense due to disease severity (38).

The study has certain limitations that should be acknowledged. Potential confounders such as prior flavivirus infection, nutritional status, and other co-morbidities were not fully controlled. Although all of the representative samples tested were positive for SARS-CoV-2 IgG, reducing intergroup variability related to COVID-19 exposure; this had no association with dengue severity. The relatively small sample size might limit the statistical power and generalizability of the findings. Although plaque reduction neutralization test (PNRT) helps in confirming DENV infection, this was not performed due to sample volume constraints. In addition, although we accessed complement activation, not all complement components (e.g., C4, C5b, C6, C7, C8, and C9) were measured. Furthermore, the cross-sectional study design limits the establishment of the causal relationship between the target analyte and disease exacerbation or mitigation over the period of disease progression, warranting longitudinal follow-up investigations to capture temporospatial disease dynamics. Moreover, high-throughput omics-based approaches such as transcriptomics, proteomics, metabolomics, and other validation experiments to identify relevant disease mediators or therapeutic targets are warranted. Lastly, the limited viremia positivity among the samples and restricted serotyping constrain the generalizability of serotype-dependent complement alterations. Taken together, these limitations suggest that the current findings should rather be considered as indicative, and further large-scale, longitudinal investigations encompassing all of the complement factors are warranted to validate the findings.

In conclusion, our investigation reinforces the importance of the complement system in dengue pathogenesis, with particular emphasis on the early classical pathway of complement activation. Furthermore, the association of sCR1 indicates its likelihood for use as a potential candidate biomarker of dengue severity, which, however, remains to be confirmed using longitudinal cohort studies. Given the strong positive correlation of serum uric acid with blood platelet counts, the relevance of uric acid (an endogenous antioxidant) can be elaborately studied in dengue. This study offers several avenues for future translational research aimed at improving dengue diagnostic, highlighting the complement factors’ potential utility as biomarker candidates in distinguishing disease severity or predicting clinical progression. Future studies incorporating longitudinal sampling, comprehensive profiling of terminal pathways, and larger, virologically diverse cohorts will be essential to validate the pathways explored herein for rendering translational validations in clinical dengue infection.