Objective: Dengue fever has been one of the most important health problems in Taiwan since a large outbreak during 1987 and 1988. It is critically necessary to have a diagnostic approach that can detect early infections in an outbreak or even find infections existing in silent transmission of the disease.

Methods: To develop an efficient diagnostic protocol, 105 plasma/serum and 35 peripheral blood leukocyte (PBL) specimens from the 1994 outbreak in southern Taiwan were collected for assessment by various diagnostic techniques in this study.

Results: In acute blood samples, dengue viruses were isolated from 19.4% (14/72) and 33.3% (14/42) of reported and confirmed cases, respectively. Viral RNA in serum/plasma was detected from 20.0% (12/60) of acute samples, which was significantly higher than that from convalescent samples (3/44; 6.8%). However, viral RNA in PBLs, detected by reverse transcription polymerase chain reaction (PBL-RT-PCR), could be observed in 73.2% (19/26) and 66.7% (6/9) of acute and convalescent samples, respectively. The persistence of dengue viruses in PBLs was also evidenced by the presence of viral antigens in 42.9% (4/7) of confirmed convalescent samples by the immunofluorescence antibody test. In addition, IgM antibodies were detected in 43.8% (46/105) of reported cases and 85.2% (46/54) of confirmed cases by the IgM antibody capture enzyme-linked immunosorbent assay (MAC-ELISA).

Conclusions: Although IgM antibody detection achieved the highest detection rate among techniques assessed in this study, no individual test can actually reach full efficiency for early diagnosis of dengue infections. Here, we propose a protocol which applies MAC-ELISA and PBL-RT-PCR in sequence, by which 22 confirmed cases were definitely proved as dengue positive. High levels of both sensitivity and specificity were shown in this protocol.