Background Vibrio cholerae is the cause of cholera, a severe watery diarrhea. Protection against cholera is serogroup specific. Serogroup specificity is defined by the O-specific polysaccharide (OSP) component of lipopolysaccharide (LPS).MethodologyHere we describe a conjugate vaccine for cholera prepared via squaric acid chemistry from the OSP of V. cholerae O1 Inaba strain PIC018 and a recombinant heavy chain fragment of tetanus toxin (OSP:rTTHc). We assessed a range of vaccine doses based on the OSP content of the vaccine (10-50 μg), vaccine compositions varying by molar loading ratio of OSP to rTTHc (3:1, 5:1, 10:1), effect of an adjuvant, and route of immunization.Principle FindingsImmunized mice developed prominent anti-OSP and anti-TT serum IgG responses, as well as vibriocidal antibody and memory B cell responses following intramuscular or intradermal vaccination. Mice did not develop anti-squarate responses. Intestinal lamina proprial IgA responses targeting OSP occurred following intradermal vaccination. In general, we found comparable immune responses in mice immunized with these variations, although memory B cell and vibriocidal responses were blunted in mice receiving the highest dose of vaccine (50 μg). We found no appreciable change in immune responses when the conjugate vaccine was administered in the presence or absence of immunoadjuvant alum. Administration of OSP:rTTHc resulted in 55% protective efficacy in a mouse survival cholera challenge model.ConclusionWe report development of an Inaba OSP:rTTHc conjugate vaccine that induces memory responses and protection against cholera in mice. Development of an effective cholera conjugate vaccine that induces high level and long-term immune responses against OSP would be beneficial, especially in young children who respond poorly to polysaccharide antigens.

BackgroundRapid and reliable diagnostic assays for enteric (typhoid and paratyphoid) fever are urgently needed. We report the characterization of novel approach utilizing lymphocyte secretions, for diagnosing patients with enteric fever by the TPTest procedure.MethodologyTPTest detects Salmonella-specific IgA responses in lymphocyte culture supernatant. We utilized TPTest in patients with suspected enteric fever, patients with other illnesses, and healthy controls. We also evaluated simplified modifications of TPTest for adaptation in laboratories with limited facilities and equipment.Principal FindingsTPTest was positive in 39 (27 typhoid and 12 paratyphoid A) patients confirmed by blood culture and was negative in 74 healthy individuals. Among 32 individuals with other illnesses, 29 were negative by TPTest. Of 204 individuals with suspected enteric fever who were negative by blood culture, 44 were positive by TPTest and the patients were clinically indistinguishable from patients with confirmed bacteremia, except they were more likely to be under 5 years of age. We evaluated simplifications in TPTest, including showing that lymphocytes could be recovered using lysis buffer or buffy coat method as opposed to centrifugation, that incubation of cells at 37°C did not require supplemental CO2, and that results were available for majority of samples within 24 hours. Positive results by TPTest are transient and revert to negative during convalescence, supporting use of the test in endemic areas. The results can also be read using immunodot blot approach as opposed to ELISA. Since no true gold standard currently exists, we used a number of definitions of true positives and negatives. TPTest had sensitivity of 100% compared to blood culture, and specificity that ranged from 78–97% (73–100, 95% CI), depending on definition of true negative.ConclusionThe TPTest is useful for identification of patients with enteric fever in an endemic area, and additional development of simplified TPTest is warranted.

BackgroundThere is an urgent need for an improved diagnostic assay for typhoid fever. In this current study, we compared the recently developed TPTest (Typhoid and Paratyphoid Test) with the Widal test, blood culture, and two commonly used commercially available kits, Tubex and Typhidot.MethodologyFor analysis, we categorized 92 Bangladeshi patients with suspected enteric fever into four groups: S. Typhi bacteremic patients (n = 28); patients with a fourfold change in Widal test from day 0 to convalescent period (n = 7); patients with Widal titer ≥1:320 (n = 13) at either acute or convalescent stage of disease; and patients suspected with enteric fever, but with a negative blood culture and Widal titer (n = 44). We also tested healthy endemic zone controls (n = 20) and Bangladeshi patients with other febrile illnesses (n = 15). Sample size was based on convenience to facilitate preliminary analysis.Principle findingsOf 28 S. Typhi bacteremic patients, 28 (100%), 21 (75%) and 18 (64%) patients were positive by TPTest, Tubex and Typhidot, respectively. In healthy endemic zone controls, the TPTest method was negative in all, whereas Tubex and Typhidot were positive in 3 (15%) and 5 (25%), respectively. We then estimated sensitivity and specificity of all diagnostic tests using Bayesian latent class modeling. The sensitivity of TPTest, Tubex and Typhidot were estimated at 96.0% (95% CI: 87.1%-99.8%), 60.2% (95% CI: 49.3%-71.2%), and 59.6% (95% CI: 50.1%-69.3%), respectively. Specificity was estimated at 96.6% (90.7%-99.2%) for TPTest, 89.9% (79.6%-96.8%) for Tubex, and 80.0% (67.7%-89.7%) for Typhidot.ConclusionThese results suggest that the TPTest is highly sensitive and specific in diagnosing individuals with typhoid fever in a typhoid endemic setting, outperforming currently available and commonly used alternatives.

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.