Demographic characteristics of study participants.

<p>Demographic characteristics of study participants.</p

Immune responses to V. cholerae (O1 Ogawa and O1 Inaba) following vaccination with BivWC.

<p>Immune responses to V. cholerae (O1 Ogawa and O1 Inaba) following vaccination with BivWC.</p

Correlation between Day 7 anti-OSP and anti-LPS ASC Responses.

<p>Ogawa: Spearman’s r = 0.7706, p < 0.0001. Inaba: Spearman’s r = 0.9030, p < 0.0001.</p

Ogawa and Inaba O-specific polysaccharide (OSP) Immunoglobulin A (IgA) Antibody secreting cell (ASC) responses in vaccinees.

<p>Individual and mean circulating antigen-specific IgA responses to Ogawa and Inaba OSP with standard error bars. P values are indicated where statistically significant differences from baseline (Day 0) are present.</p

Vibriocidal Responses.

<p>Geometric mean titer (+95% CI) of vibriocidal response to <i>V</i>. <i>cholerae</i> O1 Ogawa (A) and Inaba (B) at baseline (day 0) and 7 days after each immunization (day 7 and 21). Statistically significant differences relative to baseline are indicated (** = P < 0.01, *** = P < 0.001, **** = P < 0.0001).</p

Dried Blood Spots for Measuring <i>Vibrio cholerae</i>-specific Immune Responses

<div><p>Background</p><p><i>Vibrio cholerae</i> causes over 2 million cases of cholera and 90,000 deaths each year. Serosurveillance can be a useful tool for estimating the intensity of cholera transmission and prioritizing populations for cholera control interventions. Current methods involving venous blood draws and downstream specimen storage and transport methods pose logistical challenges in most settings where cholera strikes. To overcome these challenges, we developed methods for determining cholera-specific immune responses from dried blood spots (DBS).</p><p>Methodology/principal findings</p><p>As conventional vibriocidal assay methods were unsuitable for DBS eluates from filter paper, we adopted a drop-plate culture method. We show that DBS collected from volunteers in South Sudan, and stored for prolonged periods in field conditions, retained functional vibriocidal antibodies, the titers of which correlated with paired serum titers determined by conventional spectrophotometric methods (r = 0.94, p = 0.00012). We also showed that eluates from DBS Serum Separator cards could be used with conventional spectrophotometric vibriocidal methods, and that they correlated with paired serum at a wide range of titers (r = 0.96, p<0.0001). Similarly, we used ELISA methods to show that <i>V</i>. <i>cholerae</i> O-specific polysaccharide antibody responses from DBS eluates correlated with results from paired serum for IgG (r = 0.85, p = 0.00006), IgM (r = 0.79, p = 0.00049) and IgA (r = 0.73, p = 0.0019), highlighting its potential for use in determination of isotype-specific responses. Storage of DBS cards at a range of temperatures did not change antibody responses.</p><p>Conclusion</p><p>In conclusion, we have developed and demonstrated a proof-of-concept for assays utilizing DBS for assessing cholera-specific immune responses.</p></div

Determination of vibriocidal titers from dried blood spots collected using Whatman 903 protein (WPS) saver cards and drop-plate culture method.

<p>A. Representative image of vibriocidal titer obtained by drop-plate method using eluate from DBS WPS card obtained from a recipient of an oral cholera vaccine in South Sudan (with a known serum titer of 1280). B. Spearman correlation of vibriocidal titers determined using eluates from DBS WPS cards containing blood collected from recipients of an oral cholera vaccine in South Sudan, by drop-plate culture method with titers obtained from paired serum using conventional spectrophotometric method. C. Spearman correlation of vibriocidal titers determined using eluates from DBS WPS cards containing blood spiked with mAbA2 IgG, by drop-plate culture method with titers obtained from paired serum using conventional spectrophotometric method.</p

Two Cholkit dipsticks showing characteristic negative (left image) and positive (right image) results after 15 min sample run.

<p>Two Cholkit dipsticks showing characteristic negative (left image) and positive (right image) results after 15 min sample run.</p

Characteristics of study participants.

<p>Characteristics of study participants.</p

Comparison of RDTs with microbiological culture and PCR in 76 study participants in Bangladesh.

<p>Comparison of RDTs with microbiological culture and PCR in 76 study participants in Bangladesh.</p