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

Results of the enriched RDT and of culture at National Public Health Laboratory, Juba, and at Institut Pasteur, Paris, compared to PCR results.

<p>Results of the enriched RDT and of culture at National Public Health Laboratory, Juba, and at Institut Pasteur, Paris, compared to PCR results.</p

Diagnostic performance of direct and enriched RDT, and of culture at National Public Health Laboratory, Juba, and at Institut Pasteur, Paris, using PCR as the reference standard in all (N = 101) or patients without prior antibiotics (N = 80).

<p>Diagnostic performance of direct and enriched RDT, and of culture at National Public Health Laboratory, Juba, and at Institut Pasteur, Paris, using PCR as the reference standard in all (N = 101) or patients without prior antibiotics (N = 80).</p