Detection of dengue virus RNA in patients after primary or secondary dengue infection by using the TaqMan automated amplification system

Abstract

In consecutive serum samples from 25 tourists with acute dengue fever, virus-specific RNA was detected by using fully automated TaqMan reverse transcriptase PCR. For this amplification technique new primers and special fluorochrome-labeled probes had to be synthesized. During amplification the increasing amount of viral DNA could simultaneously be measured in the tightly sealed tubes. Dengue virus RNA was found in almost all patients (17 of 18), if the samples had been taken soon after the onset of symptoms and before anti-dengue virus antibody had been produced. RNA was detectable in only one of five persons who had anti-dengue virus immunoglobulin M (IgM) antibodies but not yet IgG antibodies. In 30 late samples with both IgG and IgM antibodies viral RNA was no longer demonstrable. In two early samples from two frequent travelers obtained 1 and 2 days after the onset of symptoms significant IgG antibody titers were present but there were no anti-dengue virus IgM antibodies. In these samples a viral load of >5 x 10(6) dengue virus RNA copies (dengue types 1 and 2) was detectable. These findings of a high viral load in the presence of anti-dengue virus IgG antibody are suggestive of a secondary dengue virus infection. In the 20 tourists (17 plus 1 plus 2) in whom viral RNA was found, the dengue virus serotype could be related to the area where the infection had taken place. Most of our patients came from southeast Asia and most frequently had dengue virus type 1 infections (8 of 20).

FIG. 1

Standardization of dengue virus type 2 TaqMan RT-PCR. Totals of 500 to 5 × 10⁶ RNA molecules/ml were diluted in human sera from healthy subjects without anti-dengue virus antibodies. A minimum of 500 RNA molecules/ml, corresponding to two to three molecules in 5 μl of RNA extract in the amplification mix, could be detected after 38 cycles. On the y axis is shown the intensity of fluorescence (delta Rn) (FAM-TAMRA), and on the x axis is shown the number of cycles.

FIG. 2

Kinetics of IgG antibody response in consecutive serum samples from nine representative patients. Each sample is represented by a circle. In addition to the IgG titers (y axis) a positive PCR is indicated by a black circle and a positive anti-dengue virus IgM response is indicated by a striped circle. Absence of IgM or RNA is indicated by a white circle. Patients 4, 12, and 21 had dengue virus type 1 infections; patients 1, 6, 9, 11, and 17 had type 2 infections; and patient 7 had a type 3 infection.