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An essential requirement related to treatment of dengue-infected patients is a rapid and accurate detection of dengue virus during febrile stage of the disease.The study examined using direct immunofluorescence staining the presence of dengue viral antigen in peripheral blood mononuclear cells (PBMCs).Four hundred forty-five blood samples from 164 patients with dengue fever, dengue hemorrhagic fever, and other febrile illnesses were collected daily from the day of admission until discharge and also at convalescent stage. Blood smear was stained with fluorescein isothiocyanate-conjugated polyvalent dengue antiserum and examined under a fluorescent microscope. Dengue infection was confirmed by virus isolation and/or dengue-specific IgM and IgG enzyme linked immunosorbent assay test.Dengue viral antigens were found in most PBMC samples of dengue infected patients collected on the day before defervescence and continued until 2 to 3 days afterward. The number of dengue viral antigen positive PBMCs was highest in patients with dengue shock syndrome. Sensitivity and specificity of this method during the febrile stage was 93.8% (95% confidence interval: 88.8%–98.9%) and 100%, respectively. The positive and negative predictive values were 100% and 69.0%, respectively.Detection of dengue viral antigen in direct PBMC smear provides a useful and rapid technique for early diagnosis of dengue virus infection.