Inter-laboratory comparison.

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.

在登革热流行且资源匮乏的地区，精准及时的诊断对于开展有效的临床管理与疫情防控至关重要，尤其是在多种虫媒病毒共同循环传播且临床表现存在重叠的场景中。然而，此类地区的绝大多数登革热诊断依赖于灵敏度有限的手段，例如临床评估，或是易于部署的方法如酶联免疫吸附试验（ELISA）与快速诊断检测（RDTs）。诊断性能更优的分子诊断技术，却因存在公认的后勤与操作壁垒，极少在参考实验室之外的场所开展应用。

本研究提供了真实世界证据，对比了在去中心化场景下用于登革热诊断的临床、血清学与分子检测手段的性能表现。2023至2024年登革热暴发期间，研究团队在秘鲁尤里马瓜斯的圣赫马医院前瞻性入组了271名急性发热患者。所有患者均接受了临床评估（采用世界卫生组织（WHO）2009年登革热分类标准），以及实验室检测：包括NS1/IgM快速诊断检测与酶联免疫吸附试验、针对寨卡病毒（ZIKV）/登革病毒（DENV）/基孔肯雅病毒（CHIKV）的三重逆转录聚合酶链反应（ZDC-PCR）、新开发的针对寨卡病毒（ZIKV）/黄热病毒（YFV）/登革病毒（DENV）/基孔肯雅病毒（CHIKV）的多重逆转录聚合酶链反应（ZYDC-PCR），以及作为金标准的血清型特异性登革热逆转录聚合酶链反应。

本研究采用灵敏度、特异度、受试者工作特征曲线下面积（ROC-AUC）分析以及逻辑回归模型，对各项诊断手段的性能进行了评估。研究选取131份样本子集，在区域实验室（尤里马瓜斯）与中央实验室（利马）之间开展了ZYDC-PCR的实验室间比对。在271例登革热疑似病例中，有88例（32.6%）经参考PCR检测确认感染。ZYDC-PCR与参考检测方法一致性极佳（灵敏度为86.0%，科恩kappa值为0.893），且在中央实验室与区域实验室中均表现出稳定的性能。基于NS1的检测手段特异度较高（≥96%），但灵敏度中等（约72%）。受试者工作特征曲线分析证实，聚合酶链反应（PCR）的诊断准确性优异（AUC=0.97），其性能优于快速诊断检测、酶联免疫吸附试验（AUC=0.85~0.89）以及临床评估（AUC=0.65）。

本研究证实，在区域医院部署多重PCR检测技术具备附加价值与可行性，可显著提升诊断准确性，实现对感染状态的更早甄别，这对于临床管理与疫情应对均至关重要。