# Assessing the diagnostic performance of clinical, serological and molecular approaches to improve dengue case detection in the Peruvian Amazon

## TL;DR

This study compares different dengue diagnostic methods in a remote Peruvian hospital, showing that PCR tests, especially a new multiplex version, are more accurate and feasible for use in low-resource settings.

## Contribution

The study demonstrates the feasibility and high diagnostic accuracy of a new multiplex PCR test for dengue in a decentralized, resource-limited setting.

## Key findings

- The new multiplex PCR (ZYDC-PCR) showed strong agreement with the reference test (sensitivity 86.0%) and consistent performance across laboratories.
- PCR tests outperformed rapid diagnostic tests and clinical assessments in diagnostic accuracy (AUC of 0.97 vs. 0.85–0.89 and 0.65, respectively).
- Despite logistical challenges, PCR testing proved feasible for implementation in a regional hospital in the Peruvian Amazon.

## Abstract

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.

Dengue is a viral infection spread by Aedes mosquitos that causes fever and, in some cases, can lead to severe illness. For proper patient care and outbreak control, timely and accurate case confirmation is essential. However, in many dengue endemic areas, especially in remote and low-resource settings, access to reliable laboratory tests is limited. As a result, dengue suspected cases are often diagnosed based on clinical symptoms alone or simple rapid tests, that may miss true infections. In this study, carried out in a district hospital in the Peruvian Amazon, we compared how well different diagnostic approaches work in real-world conditions. We evaluated: (1) diagnosis based on symptoms, (2) rapid tests and ELISA tests that detect the dengue protein NS1 or antibodies and (3) three different PCR tests that detect the virus’s genetic material. One of the PCRs was a new test detecting simultaneously dengue and three other arboviruses. We found that adding NS1 rapid tests or NS1 ELISA to clinical diagnosis improved dengue detection, but these tests still missed many confirmed infections. PCR testing showed highest accuracy, and the new PCR worked similar to the reference test, even when used in a small district hospital setting. Despites the encountered logistic, infrastructure and trained human resources challenges, our findings show the feasibility of PCR decentralization in a district hospital in the Peruvian Amazon.