Clinical and Environmental Surveillance for Vibrio cholerae in Resource Constrained Areas: Application During a 1-Year Surveillance in the Far North Region of Cameroon

Abstract

Biological confirmation of the presence of Vibrio cholerae in clinical and environmental samples is often constrained due to resource- and labor-intensive gold standard methods. To develop low-cost, simple, and sustainable surveillance techniques, we modified previously published specimen sampling and culture techniques and applied the use of enriched dipstick testing in conjunction with the use of filter paper for DNA specimen preservation during clinical and environmental surveillance in the Far North of Cameroon from August 2013 to October 2014. The enriched dipstick methodology during routine use in a remote setting demonstrated a specificity of 99.8% compared with polymerase chain reaction (PCR). The novel application of filter paper as a preservation method for cholera DNA specimens reduced the need for cold chain storage and allowed for PCR characterization and confirmation of V. cholerae. The application of basic technologies such as the enriched dipstick, the use of simplified gauze filtration for environmental sample collection, and the use of filter paper for sample preservation enabled early case identification with reduced logistics and supply cost while reporting minimal false-positive results. Simplified laboratory and epidemiological methodologies can improve the feasibility of cholera surveillance in rural and resource-constrained areas, facilitating early case detection and rapid response implementation.

Figure 1.

Crystal VC kit. (A) 1) The dipstick is provided in an individualized, humidity controlled package; 2) a clean test tube is provided in the kit for each dipstick tested; 3) a Pasteur pipette is provided in the kit for each specimen to be transferred; and 4) a specimen processing vial containing enrichment media. (B) 1) A clean dipstick; 2) a negative dipstick showing clearly the control line only; 3) a Vibrio cholerae O1–positive dipstick showing both the control line and the O1-positive line. (C) A V. cholerae O1–positive dipstick as it appears when testing in the kit-provided test tube; note that only ∼200 μL of specimen should be added to the tube as indicated by the arrow.

Figure 2.

Procedure for detecting Vibrio cholerae O1 from environmental source using enriched dipstick method. Figure 2 is a pictorial of the steps used to collect and assess V. cholerae O1 from environmental samples. Step 1 is to collect 2–3 L water from environmental sampling site. Step 2 shows that one should take medical gauze and fold it, then roll it into a tube to be placed into the mouth of the funnel device, ensuring that the gauze fits snug into the mouth of the funnel so that it will not be displaced when funneling water. The funnel can be a plastic bottle with the base cutoff or, if available, a funnel. Once the gauze filter is in place in the mouth of the funnel, then 2–3 L water is filtered through the funnel. Step 3 is once the water has all been filtered through the funnel, remove the gauze filter and place into a 50-mL conical tube containing 20 mL of 2× alkaline peptone water (APW). The APW is 2× to offset any water retained in the filter, which would reduce the concentration of the APW. Step 4 is to incubate the gauze in the 2× APW solution for ∼24 hours at ∼37°C (or between 25°C and 40°C room temperature [RT] if incubator is not available). Step 5 is to use the Pasteur pipette included in the Crystal VC dipstick kit and transfer ∼200 μL of the enriched media to the test tube provided with the kit. Then insert a new dipstick into the solution and allow to incubate at RT for 15 minutes. Read the dipstick at 15 minutes to determine if it is positive or negative (see Figure 1B above). Step 6 is to preserve the specimen in Cary Blair and on precut individual Whatman filter paper cards; all positives and a 10% sample of negatives were preserved on Cary Blair and filter paper for culture confirmation.

Figure 3.

Monthly enrollments of diarrhea and cholera cases. The graph above shows the enrollment of diarrheal cases by month, stratified by surveillance type and cholera outcome.

Figure 4.

Percentage of diarrhea cases with severe dehydration stratified by age group across health facilities. The graph demonstrates the percentage of severe diarrheal cases enrolled at each health facility participating in the surveillance efforts. Blangoua had the highest numbers among all age groups.