Defining the clinical utility of dengue RT-iiPCR: stratification by days post-onset and primary vs. secondary...

Rapid molecular diagnostics are critical for timely dengue virus (DENV) detection, especially in resource-limited settings. Reverse transcription-insulated isothermal polymerase chain reaction (RT-iiPCR) enables sample-to-answer amplification without thermocyclers. While this platform was previously validated, its diagnostic performance against contemporary viral genotypes and, critically, across different host immune statuses (primary vs. secondary infection) remains largely undefined. We assessed a DENV iiPCR assay against quantitative real-time reverse transcription PCR (qRT-PCR) during the 2023 outbreak in southern Taiwan.

Acute-phase sera (n = 185) from febrile patients were tested by qRT-PCR, pan-DENV iiPCR, IgM/IgG ELISAs, and serotype-specific RT-PCR/iiPCR. Diagnostic performance was evaluated with qRT-PCR as a reference. Subgroup analyses examined effects of days post-symptom onset (PSO), immune status (primary vs secondary infection), and serotype on iiPCR sensitivity.

Pan-iiPCR achieved 88.4 % sensitivity and 100 % specificity versus qRT-PCR. Sensitivity peaked within 3 days PSO (96.8 %) and in primary infections (95.0 %) but declined after day 3 (53.3 %) and in secondary infections (82.2 %). Combining pan-iiPCR with IgM testing raised sensitivity to ≥99 % beyond the viremic window. iiPCR correctly typed 83.5 % (71/85) of DENV-1 and 94.3 % (66/70) of DENV-2. Because pan-iiPCR served as the screening step, 18 RT-PCR-positive specimens were pan-iiPCR negative and therefore did not proceed to iiPCR serotyping.

iiPCR enables rapid early-phase detection and is useful when paired with serology. However, reduced sensitivity in low-viremia and secondary infections, as well as incomplete serotyping, limit standalone use. Primer optimization and integration with IgM testing may enhance outbreak utility.