Recognizing cholera cases early, especially in the initial phase of an outbreak and in areas where cholera has not previously circulated, is a high public health priority. Laboratory capacity in such settings is often limited. To address this, we have developed a rapid diagnostic test (RDT) termed Cholkit that is based on an immunochromatographic lateral flow assay for the diagnosis of cholera cases using stool. Cholkit contains a monoclonal antibody (ICL-33) to the O-specific polysaccharide (OSP) component of V. cholerae O1 lipopolysaccharide, and recognizes both Inaba and Ogawa serotypes. We tested the Cholkit dipstick using fresh stool specimens of 76 adults and children presenting with acute watery diarrhea at the icddr,b hospital in Dhaka, Bangladesh. We compared Cholkit’s performance with those of microbial culture, PCR (targeting the rfb and ctxA genes of V. cholerae) and the commercially available RDT, Crystal VC (Span Diagnostics; Surat, India). We found that all stool specimens with a positive culture for V. cholerae O1 (n = 19) were positive by Cholkit as well as Crystal VC. We then used Bayesian latent class modeling to estimate the sensitivity and specificity of each diagnostic assay. The sensitivity of Cholkit, microbiological culture, PCR and Crystal VC was 98% (95% CI: 88–100), 71% (95% CI: 59–81), 74% (95% CI: 59–86) and 98% (95% CI: 88–100), respectively. The specificity for V. cholerae O1 was 97% (95% CI: 89–100), 100%, 97% (95% CI: 93–99) and 98% (95% CI: 92–100), respectively. Of note, two Crystal VC dipsticks were positive for V. cholerae O139 but negative by culture and PCR in this area without known circulating epidemic V. cholerae O139. In conclusion, the Cholkit dipstick is simple to use, requires no dedicated laboratory capacity, and has a sensitivity and specificity for V. cholerae O1 of 98% and 97%, respectively. Cholkit warrants further evaluation in other settings. Cholera is a severely dehydrating diarrheal disease that can lead to death if remains untreated. The incidence of case fatality is higher at the beginning of the outbreak. Diagnosis of cholera in the early stage of outbreak is a high public health priority. Although countries facing complex emergencies are more vulnerable to cholera outbreak, laboratory capacity in such settings is usually limited. To address this, here we report the development of a rapid diagnostic test (RDT) termed Cholkit for the diagnosis of cholera cases using stool and the assessment of its performance with those of microbial culture, PCR and Crystal VC assay, a commercially available dipstick using a latent class modeling approach.

Cholera is an acute voluminous dehydrating diarrheal disease caused by toxigenic strains of Vibrio cholerae O1 and occasionally O139. A growing body of evidence indicates that immune responses targeting the O-specific polysaccharide (OSP) of V. cholerae are involved in mediating protection against cholera. We therefore assessed whether antibody responses against OSP occur after vaccination with live attenuated oral cholera vaccine CVD 103-HgR, and whether such responses correlate with protection against cholera. We assessed adult North American volunteers (n = 46) who were vaccinated with 5 × 10⁸ colony-forming units (CFU) of oral cholera vaccine CVD 103-HgR and then orally challenged with approximately 1 × 10⁵ CFU of wild-type V. cholerae O1 El Tor Inaba strain N16961, either 10 or 90 days post-vaccination. Vaccination was associated with induction of significant serum IgM and IgA anti-OSP and vibriocidal antibody responses within 10 days of vaccination. There was significant correlation between anti-OSP and vibriocidal antibody responses. IgM and IgA anti-OSP responses on day 10 following vaccination were associated with lower post-challenge stool volume (r = −0.44, P = 0.002; r = −0.36, P = 0.01; respectively), and none of 27 vaccinees who developed a ≥1.5 fold increase in any antibody isotype targeting OSP on day 10 following vaccination compared to baseline developed moderate or severe cholera following experimental challenge, while 5 of 19 who did not develop such anti-OSP responses did (P = 0.01). Oral vaccination with live attenuated cholera vaccine CVD 103-HgR induces antibodies that target V. cholerae OSP, and these anti-OSP responses correlate with protection against diarrhea following experimental challenge with V. cholerae O1. ClinicalTrials.gov NCT01895855 Cholera is a severe watery diarrheal disease, caused by pathogenic strains of V. cholerae. Protective immunity against cholera is serogroup specific, and serogroup specificity is determined by the O-specific polysaccharide (OSP) of V. cholerae lipopolysaccharide (LPS). Despite this, no previous work has directly assessed correlation of OSP-immune responses and protection against cholera. In this study, we assessed adult North American volunteer’s antibody responses targeting OSP after vaccination with live attenuated oral cholera vaccine CVD 103-HgR, and we assessed correlation of protection against cholera with such antibody responses. Oral vaccination was associated with the induction of significant IgM and IgA responses against OSP, and these responses correlated with vibriocidal responses. There was significant negative correlation of OSP responses and post-challenge stool volume, and none of the volunteers who developed an anti-OSP antibody responses of any isotype of ≥1.5 fold developed moderate or severe cholera following experimental challenge. In summary, vaccination with live attenuated oral cholera vaccine CVD 103-HgR induces antibodies that target V. cholerae OSP, and these responses highly correlate with protection against cholera.