<p>Inter-laboratory comparison.</p>

In dengue endemic, resource-limited settings, accurate and timely diagnosis is critical for effective clinical management and outbreak control, especially where multiple arboviruses co-circulate and overlap in clinical presentations. However, most dengue diagnosis in such settings rely on approaches with limited sensitivity such as clinical assessment, or easily deployable methods such as ELISA or rapid diagnostic tests (RDTs). Molecular diagnostics with superior diagnostic performance are rarely implemented beyond reference laboratories due to perceived logistical and operational barriers. This study provides real-world evidence comparing the performance of clinical, serological and molecular approaches for dengue diagnosis in a decentralized setting. We prospectively enrolled 271 patients with acute febrile illness at Santa Gema Hospital in Yurimaguas, Peru, during a dengue outbreak in 2023–2024. Patients underwent clinical evaluation (WHO 2009 dengue classification), and laboratory testing including NS1/ IgM RDTs and ELISAs, a triplex RT-PCR for ZIKV/DENV/CHIKV (ZDC-PCR), a newly developed multiplex RT-PCR for ZIKV/YFV/DENV/CHIKV (ZYDC-PCR), and a serotype-specific dengue RT-PCR used as reference. Diagnostic performance was assessed using sensitivity, specificity, ROC-AUC analysis, and logistic regression models. A subset of 131 samples underwent inter-laboratory comparison of the ZYDC-PCR between the regional (Yurimaguas) and central (Lima) laboratories. Of the 271 dengue-suspected cases, 88 (32.6%) were confirmed by the reference PCR. The ZYDC-PCR had a strong agreement with the reference (sensitivity 86.0%, Cohen’s kappa 0.893) and consistent performance across the central and regional laboratory. NS1-based tests showed high specificity (≥96%) but moderate sensitivity (~72%). ROC analysis confirmed the accuracy of PCR (AUC = 0.97), outperforming RDTs, ELISAs (AUC = 0.85 to 0.89) and clinical assessment (AUC = 0.65). Our study demonstrates the added value and feasibility of implementing a multiplex PCR at a regional hospital to significantly improve diagnostic accuracy, enabling earlier detection of disease presence or absence, critical for clinical management and outbreak response.