Fig. 3. Applying the rapid test to analyze human patient sera.

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Fig. 3. Applying the rapid test to analyze human patient sera. Applying the rapid test to analyze human patient sera. (A) Map showing the endemic virus regions where the rapid tests were deployed to analyze patient serum samples. The areas of the circles correlate with the numbers of samples analyzed. The blue colors, faint to dark, represent DENV1–4. ZIKV is indicated in orange color. (B) ELISA results showing the amounts of DENV NS1 (left) and ZIKV NS1 (right) found in patient serum and supernatants from infected cell cultures. Lanes 1 and 6 are supernatants from Vero cells infected with DENV; lanes 2 and 7 are supernatants from Vero cells infected with ZIKV. Lanes 3 and 8 are PCR-negative sera; lanes 4 and 9 are sera from PCR-positive DENV patients. Lanes 5 and 10 are sera from PCR-positive ZIKV patients. (C) Images of rapid test analysis of DENV NS1 serotypes 1 to 4 and ZIKV NS1 on serotype-specific strips 1 to 4, as well as pan-DENV (P) and ZIKV (Z); the upward arrows mark positive tests, and θ is serum from an uninfected patient. (D to G) Quantification of rapid test results. Dipstick tests were run with PCR-confirmed DENV sera or ELISA-validated ZIKV serum (C), and the resulting signals were quantified and expressed as box plots. Statistical significance, based on one-way analysis of variance (ANOVA), is indicated as ***P < 0.001. (H) Statistical significance, based on an unpaired t test, is presented as *P < 0.05. In the box-and-whisker plots, the black × represents the maximum and minimum measured normalized intensity values, whereas the small square box (□☐) represents the mean value, and the larger box represents the 25 to 75% range of the data. Individual colored points represented individual patient samples measured. (I and J) Images of rapid tests showing that DENV and ZIKV NS1 tests do not cross-react. (I) Supernatants from Vero cells infected with DENV4 were chromatographed on DENV serotype strips 1 to 4 on the pan-DENV strip (P) and on the ZIKV NS1 strip (Z). (J) Supernatants from Vero cells infected with ZIKV were chromatographed on DENV serotype strips 1 to 4, on the pan-DENV strip (P), and on the ZIKV NS1 strip (Z). (K) Images of rapid tests showing ZIKV NS1 are detected in serum samples concentrated five times, but ZIKV NS1 is not detected in concentrated urine. Three sets of paired serum and urine samples were concentrated five times by filter centrifugation and chromatographed on the ZIKV dipsticks. S, serum; U, urine. (I to K) The red boxes and vertical black lines serve as fiducial markers for image recognition and processing. Upward arrows indicate positive tests using the serum samples. (L to N) Quantification of NS1 protein in supernatants of Vero cells infected separately with three DENV4 patient isolates (L) or three ZIKV patient isolates (M) or five paired serum/urine patient samples (N). (L to N) One-way ANOVA was used to calculate statistical significance of the dengue and Zika tests: ***P < 0.001. In the box-and-whisker plots, the black × represents the maximum and minimum measured normalized intensity values, whereas the black box (□) represents the mean value, and the larger box represents the 25 to 75% range of the data. Individual colored points represented individual patient samples measured. Irene Bosch et al., Sci Transl Med 2017;9:eaan1589 Published by AAAS