⦁ Diarrhea cases (age ≥2 months): Any patient attending hospital with 3 or more loose or liquid stools within 24 hours or 3 loose/liquid stools or fewer causing dehydration in the last 24 hours.
Surveillance
Surveillance teams were established with a physician, a nurse, a medical technologist, and a trained field attendant. At each site, an assigned nurse prepared a daily list of patients with diarrhea (both inpatient and outpatient). Four patients with diarrhea who met the case definition and had no other severe comorbidity (eg, severe acute respiratory illness, acute cardiovascular symptoms, or severe acute neurological disorder) were enrolled by the physician from Saturday to Wednesday each week. Two patients with diarrhea aged less than 5 years old and 2 patients aged 5 years or older were enrolled each day; if the target number of patients in a particular age group was not met, we overenrolled in the other group to meet the target of 4 patients. Upon receiving consent, the physician collected the patient’s sociodemographic characteristics such as age, gender, profession (eg, service holders such as those working in banks, schools, public or private organizations or workers paid/contracted on a daily basis involved in manual labor, construction, or agriculture), diet history, medical history (including assessment of dehydration status), sanitation, and hygiene information; and a stool sample was collected for testing. The specimens were transported in Cary-Blair transport media within 15 days to the laboratories in Dhaka at the icddr,b, and IEDCR. Specimens were immediately processed in the laboratory. Stool samples were analyzed for detection/presence of V. choleare O1/ O139.
Laboratory Procedures
For the identification of V. cholerae, specimens were streaked onto taurocholate-tellurite gelatin agar (TTGA) and incubated overnight at 37°C. Specimens were also inoculated in alkaline peptone water for enrichment and incubated for an additional 18–24 hours \citep{Bwire2017} and plated on TTGA. Slide agglutination tests performed with polyvalent and specific antisera (Denka Seiken, Japan) were carried out to differentiate the Ogawa and Inaba serotypes. Agglutination of chicken erythrocytes and polymyxin B susceptibility tests were carried out to confirm biotypes \citep{Rahman1987}\citep{Chowdhury2015}.