Evaluation of RT-PCR as a tool for diagnosis of secondary dengue virus infection

Dengue fever and dengue hemorrhagic fever are serious illnesses in many tropical and subtropical countries. Laboratory tests are essential for the confirmation of dengue virus infection. In the present study, we examined the reliability of reverse transcriptase polymerase chain reaction (RT-PCR) in the laboratory diagnosis of dengue, especially in secondary dengue virus infections. We defined the day when fever subsided as fever day 0. In primary dengue virus infection, the dengue viral genome was detected in all of the 7 samples which were collected on fever day -1 or earlier, in 3 of 4 samples on fever day 0, and in 1 of 2 samples on fever day 1. None of the samples collected on fever day 2 or later were positive by RT-PCR. In secondary dengue virus infection, the dengue viral genome was detected in all of the 28 samples which were collected on fever day -2 or earlier, in 25 of 26 on fever day -1, in 29 of 34 on fever day 0, and in 5 of 10 on fever days 1-2. None of the samples collected on fever day 3 or later were positive. Virus isolation and direct titration were attempted using the plasma samples. When the data of secondary infection cases were analyzed based on fever day, dengue viruses were isolated from all of the 5 samples which were collected on fever day -2 or earlier, in 5 of 13 samples on fever day -1, and in 4 of 22 on fever day 0, but were not isolated from any of the 4 samples collected on fever days 1-2. Viruses were directly detected in 7 of 11 samples on fever day -2 or earlier, in 4 of 13 on fever day -1, and in 1 of 16 on fever day 0. These results indicate that RT-PCR is more sensitive than virus isolation and direct virus titration for determining secondary dengue virus infection. The results also suggest that RT-PCR is a useful diagnostic test for confirmation of dengue virus infection in secondary infection as well as in primary infection, especially when plasma samples are collected before the fever subsides.