The plate was incubated at 37°C for 1 hour. After washing six times, the plates were incubated at 37°C following the addition of HRP-conjugated anti-NS1 mAb. The wells were then washed six times and TMB was added to the wells. The plates were further incubated at room temperature for 10 minutes in the dark. The reaction was stopped with stop solution (1 M H3PO4) and the plates were read. The index value was determined by dividing the average OD of each sample by the cut-off value. The cut-off value was determined by multiplying the average OD of the calibrator by the calibration factor provided

by the manufacturer. Index values of <0.9, 0.9–1.1, and >1.1 were considered negative, equivocal, and positive, respectively. Equivocals were regarded as negative.[14, 17] Tabulation, management, and analysis of raw data were performed using Microsoft Excel. Detection rates were determined AZD1208 price as reported previously.[18, 19] Study outcomes were compared by chi-squared test and Fisher’s exact test (for small sample sizes with n < 10), using the QI Macros statistical analysis program (KnowWare International, Denver, CO, USA). A total of 484 serum samples collected from 2007 to 2011 were used in the study. In 336 serum samples DENV infection was confirmed; 301 (90%) were found to be NS1 positive using the Biorad assay. All 148 serum samples from confirmed Fulvestrant mouse dengue-negative

donors yielded negative results. Of 336 samples tested by both the NS1 antigen ELISA and RT-PCR, 223 (66%) were positive by both the NS1 antigen ELISA and RT-PCR. A total of 78 samples (23%) were positive by NS1 antigen ELISA but negative by RT-PCR,

3 (1%) MycoClean Mycoplasma Removal Kit samples were negative by the NS1 antigen ELISA but positive by RT-PCR, and 32 samples were negative by both NS1 ELISA and RT-PCR. Detection rates between NS1 ELISA and RT-PCR were statistically significant (chi-squared test, p < 0.01). Of the 336 serum samples from DENV confirmed patients, 199 serum samples were performed in parallel with the Panbio assay. The Panbio assay identified 102 (51%) as NS1 positive of 199 serum samples from DENV infection confirmed cases. Additionally, the Panbio assay identified all 24 serum samples from confirmed dengue-negative cases as negative. The p value (chi-squared test, p < 0.01) suggests that the levels of sensitivity between the two kits were statistically different. The rate of detection using RT-PCR was 76% during the early phase (1–5 days after onset of disease) but decreased later during infection (days 6–10 = 20%, ≥11 days = 0%) (Table 1). In contrast, the rate of detection using NS1 was 93% (days 1–5) and 91% (days 6–10). As with RT-PCR, the rate of NS1 detection decreased to 50% at the later phase of infection (≥11 days) (Table 1). The rate of detection using IgM was lower (60%) as compared to RT-PCR and NS1 during the early phase of the disease (days 1–5), but was high during the later phase of disease (days 6–10 = 90% and ≥11 days = 93%, Table 2).