Flow cytometry compared with indirect immunofluorescence for rapid detection of dengue virus type 1 after amplification in tissue culture

Dengue virus (DV) was detected early in infected mosquito C6/36 cells by us ing indirect immunofluorescence (IF) in conjunction with flow cytometry. Th ree fixation-permeabilization methods and three DV serotype I (DEN-1)-speci fic monoclonal antibodies, 8-8 (anti-E), 16-4 (anti-NS1), and 15F3-1 (anti- NS1), were evaluated for the detection of DEN-1 in infected C6/36 cells. We found that these three monoclonal antibodies were capable of detecting DV in C6/36 cells as early as 24 h postinoculation by using a conventional ind irect IF stain. Both 8-8 and 16-4 detected DV earlier and showed a greater number of DV-positive cells than 15F3-1. In flow cytometry, 3% paraformalde hyde plus 0.1% Triton X-100 with 16-4, the best fixation-permeabilization m ethod for testing DV, showed higher sensitivity (up to 1 PFU) than indirect IF stain. The higher sensitivity of 16-4 in detecting DEN-1 was found with both IF and flow cytometry. Flow cytometry, which had a sensitivity simila r to that of nested reverse transcription-PCR, was more sensitive in detect ing DV in the infected mosquito cells 10 h earlier than the conventional IF stain. When clinical specimens were amplified in mosquito C6/36 cells and then assayed for DV using flow cytometry and conventional virus isolation a t day 7 postinfection, both methods had 97.22% (35 out of 36) agreement. Mo reover, among 12 positive samples which were detected by conventional cultu re method, the flow cytometry assay could detect DV in 58.33% (7 out of 12) of samples even at day 3 postinfection. In conclusion, both monoclonal ant ibodies 8-8 and 16-4 can be used for the early detection of DEN-1-infected C6/36 cells, with 16-4 (anti-NS1) being the best choice for the rapid diagn osis of DV by both the IF staining and flow cytometry methods.