Dried Blood Spots for Measuring Vibrio cholerae-specific Immune Responses.

BACKGROUND: Vibrio cholerae 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). METHODOLOGY/PRINCIPAL FINDINGS: 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 V. cholerae 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. CONCLUSION: In conclusion, we have developed and demonstrated a proof-of-concept for assays utilizing DBS for assessing cholera-specific immune responses.

Cholera Rapid Test with Enrichment Step Has Diagnostic Performance Equivalent to Culture.

Cholera rapid diagnostic tests (RDT) could play a central role in outbreak detection and surveillance in low-resource settings, but their modest performance has hindered their broad adoption. The addition of an enrichment step may improve test specificity. We describe the results of a prospective diagnostic evaluation of the Crystal VC RDT (Span Diagnostics, India) with enrichment step and of culture, each compared to polymerase chain reaction (PCR), during a cholera outbreak in South Sudan. RDTs were performed on alkaline peptone water inoculated with stool and incubated for 4-6 hours at ambient temperature. Cholera culture was performed from wet filter paper inoculated with stool. Molecular detection of Vibrio cholerae O1 by PCR was done from dry Whatman 903 filter papers inoculated with stool, and from wet filter paper supernatant. In August and September 2015, 101 consecutive suspected cholera cases were enrolled, of which 36 were confirmed by PCR. The enriched RDT had 86.1% (95% CI: 70.5-95.3) sensitivity and 100% (95% CI: 94.4-100) specificity compared to PCR as the reference standard. The sensitivity of culture versus PCR was 83.3% (95% CI: 67.2-93.6) for culture performed on site and 72.2% (95% CI: 54.8-85.8) at the international reference laboratory, where samples were tested after an average delay of two months after sample collection, and specificity was 98.5% (95% CI: 91.7-100) and 100% (95% CI: 94.5-100), respectively. The RDT with enrichment showed performance comparable to that of culture and could be a sustainable alternative to culture confirmation where laboratory capacity is limited.