Molecular Detection of Dengue Virus from Different Clinical Specimens in Suspected Dengue Patients

Abstract

Background: In our earlier seroprevalence study at this centre, dengue seropositivity was documented in 13.10% of 1076 suspected patients using NS1, IgM and IgG ELISA; however, the true burden of acute infection and the circulating viral serotypes remained undefined. This continuation study was undertaken to detect dengue virus (DENV) RNA by real-time reverse transcription polymerase chain reaction (RT-PCR) in different clinical specimens obtained from suspected dengue patients. Methods: A hospital-based prospective cross-sectional study was conducted in the Department of Microbiology, Darbhanga Medical College and Hospital, from May 2024 to April 2025. Paired serum and EDTA whole blood were collected from 250 clinically suspected dengue patients; urine (n=186) and saliva/oral fluid (n=172) were obtained wherever feasible. Serum was tested by NS1 antigen ELISA and all specimens were subjected to pan-dengue real-time RT-PCR; positive samples were serotyped by multiplex real-time RT-PCR. Results: DENV RNA was detected in 86 of 250 patients (34.4%), whereas NS1 antigen was positive in 74 (29.6%). Among the 86 molecularly confirmed cases, whole blood showed the highest RNA detection rate (97.7%), followed by serum (88.4%), urine (62.1%) and saliva (59.0%). Whole blood and serum performed best within the first five days of illness, whereas urine remained positive beyond day 7. NS1 ELISA showed a sensitivity of 82.6% and a specificity of 98.2% against RT-PCR (Cohen's kappa = 0.835). DENV-2 was the predominant serotype (46.5%), followed by DENV-3 (27.9%), DENV-1 (17.4%), DENV-4 (5.8%) and mixed infection (2.3%). Conclusion: Molecular testing markedly improves early dengue confirmation over NS1 antigen detection alone. Whole blood is the most reliable specimen during acute illness, while urine and saliva extend the diagnostic window and offer non-invasive alternatives, particularly in children and in late presenters.

Keywords: Dengue virus; real-time RT-PCR; NS1 antigen; whole blood; urine; saliva; serotyping