Application of a paper based device containing a new culture medium to detect Vibrio cholerae in water samples collected in Haiti.

Cholera is now considered to be endemic in Haiti, often with increased incidence during rainy seasons. The challenge of cholera surveillance is exacerbated by the cost of sample collection and laboratory analysis. A diagnostic tool is needed that is low cost, easy-to-use, and able to detect and quantify Vibrio cholerae accurately in water samples within 18-24h, and perform reliably in remote settings lacking laboratory infrastructure and skilled staff. The two main objectives of this study were to develop and evaluate a new culture medium embedded in a new diagnostic tool (PAD for paper based analytical device) for detecting Vibrio cholerae from water samples collected in Haiti. The intent is to provide guidance for corrective action, such as chlorination, for water positive for V. cholerae epidemic strains. For detecting Vibrio cholerae, a new chromogenic medium was designed and evaluated as an alternative to thiosulfate citrate bile salts sucrose (TCBS) agar for testing raw water samples. Sensitivity and specificity of the medium were assessed using both raw and spiked water samples. The Vibrio cholerae chromogenic medium was proved to be highly selective against most of the cultivable bacteria in the water samples, without loss of sensitivity in detection of V. cholerae. Thus, reliability of this new culture medium for detection of V. cholerae in the presence of other Vibrio species in water samples offers a significant advantage. A new paper based device containing the new chromogenic medium previously evaluated was compared with reference methods for detecting V. cholerae from spiked water sample. The microbiological PAD specifications were evaluated in Haiti. More precisely, a total of 185 water samples were collected at five sites in Haiti, June 2014 and again in June 2015. With this new tool, three V. cholerae O1 and 17 V. cholerae non-O1/O139 strains were isolated. The presence of virulence-associated and regulatory genes, including ctxA, zot, ace, and toxR, was confirmed using multiplex PCR. The three V. cholerae O1 isolates were positive for three of the four virulence-associated and regulatory genes. Twelve of the V. cholerae non-O1/O139 isolates were found to carry toxR, but none were ctxA+, zot+, or ace+. However, six of the V. cholerae non-O1/O139 isolates were resistant to penicillin, ampicillin, trimethoprim/sulfamethoxazole, nalidixic acid, and ciprofloxacin. The paper based analytical device (PAD) provides advantages in that standard culture methods employing agar plates are not required. Also, intermediary isolation steps were not required, including transfer to selective growth media, hence these steps being omitted reduced time to results. Furthermore, experienced technical skills also were not required. Thus, PAD is well suited for resource-limited settings.