# Dengue and Flavivirus Co-Infections: Challenges in Diagnosis, Treatment, and Disease Management
## 5 Implications of Better Understanding Flaviviral Co-Infections  
### Better Disease Diagnosis  
Classification of dengue based on clinical symptoms alone is complicated by the circulation of pathogens that cause similar clinical symptoms to DENV infection, such as Chikugunya virus (CHIKV) and ZIKV, which may hamper accurate descriptions of DENV dynamics [64; 65]. At present, a series of diagnostic tests have been developed for dengue, aiming at different immunological markers for virus recognition or the host immune reaction (Table 3) [72]. ELISA-based dengue assays have shown improved diagnosis accuracy, nonetheless, are still unable to provide serotype-specific identification, thereby limiting the diagnosis precision [73; 74]. For instance, DENV- specific IgM-based detection is only feasible 5-7 days post-infection. To counter the limitation, serological assays targeting DENV NS1 are innovated, offering opportunities for early dengue detection. Although the NS1-based \begin{table}
\begin{tabular}{l l} \hline \hline
**Diagnostic Test** & **Immunological Target** \\ \hline Polymerase chain reaction & RNA detection \\ Rapid tests & NS1, IgM, and IgG \\ Virus isolation & Virus \\ Immunofluorescence (IF) & Virus, IgM, and IgG \\ Plaque assay (PA) and fluorescent focus assay (FFA) & Virus titer \\ Enzyme-linked immunosorbent assay (ELISA) & NS1, IgG, IgM, and IgA \\ Neutralization test & Neutralizing antibodies (IgG) \\ \hline \hline \end{tabular}
\end{table} 
Table 3: Commonly used diagnostic tests for dengue detection. assays generate diagnosis sensitivity of approximately 80-100% and specificity of 100%, it is noteworthy that flaviviruses share high similarities in their NS1 proteins, which eventually affect the overall specificity of the tests [73; 75]. For instance, DENV-2 infections consistently show reduced NS1 detection sensitivity across multiple platforms, likely due to epitope variations and viral evolution [76; 77]. Furthermore, NS1-based assays are incapable of discerning individual serotypes involved in DENV co-infections [76].The NS1 test sensitivity is also compromised (reduced to only 39-47%) in diagnosing secondary infections due to the presence of pre-existing anti-NS1 antibodies. This phenomenon has been well observed in regions where DENV and ZIKV are co-circulating [78; 79]. False positives in NS1-based assays can occur when exposed to artificially high viral titers (>10\({}^{7}\) pfu/mL) [78]. In this light, combination of NS1 RDT, IgM ELISA, and clinical parameters has been utilized to enhance diagnostic accuracy, which improves the diagnostic sensitivity to 90.3% and specificity to 96.2% [79]. In NS1 RDT, ultrafiltration of serum samples collected at the early stages of infection is performed to improve the test sensitivity by 15-20% [80]. Several emerging multiplex platforms, such as serotype-specific NS1 lateral flow immunoassays (LFIAs), have been proposed for more accurate diagnosis due to their high result concordance with that of RT-PCR without cross-reactivity [81]. Although with high throughputs, key limitations persist in those systems, for instance, high operational costs, hence limited use in areas impacted by flavivirus outbreaks [76]. The NS1 detection usually declines after day 4-5 of illness, emphasizing the need for IgM-based assays for more accurate disease diagnosis [77]. A pooled summary of the diagnostic performance of NS1-based assays is presented in Table 4  
As aforementioned, the specificity of DENV NS1 assays is greatly affected by cross-reactivity with other flaviviruses, including WNV, JEV, and YFV, which renders false-positive results, complicating the dengue diagnosis. For instance, patients previously vaccinated or infected with other flaviviruses produce antibodies that can react with DENV antigens, thus resulting in misleading results. In terms of DENV co-infection, the majority of the tests are unable to identify the DENV serotypes, and therefore, an early prognosis of severe dengue caused by the infecting serotype is hardly made [10].