Definitions

Confirmed Cholera Case

• A suspected case with V. cholerae O1 or O139 confirmed by culture or PCR.

For countries with the necessary diagnostic capacity and where cholera was never known to be present, or has been eliminated, the V. cholerae O1 or O139 strain is demonstrated to be toxigenic.

Cholera Outbreak

• A cholera outbreak is defined by the occurrence of at least one confirmed case of cholera and evidence of local transmission.

In areas with sustained (year-round) transmission, cholera outbreaks are defined as an unexpected increase (in magnitude or timing) of suspected cases, over 2 consecutive weeks, of which some are laboratory confirmed. Such increases should be investigated and responded to appropriately through additional outbreak response and control measures.

For additional definitions, see appendix 1 – Definitions.

Laboratory Confirmation

• When a cholera outbreak is suspected and an alert is triggered, collect stool samples from symptomatic individuals and send them to the reference laboratory for microbiological confirmation by culture and/or PCR and antimicrobial susceptibility testing.

• Currently available RDTs do not replace stool culture or PCR to confirm cholera (specificity is low and therefore false positives can occur); however, if RDTs are available at the health facility, prioritize samples from patients who tested RDT-positive for laboratory confirmation.

• RDTs are used as a tool for early outbreak detection only—for triggering a cholera alert—but not to confirm the cholera outbreak. Once the outbreak is declared, RDTs  can be used for triaging the samples to be sent to the laboratory.

• For each new geographical area (district, province or region) affected by the outbreak, conduct laboratory confirmation of suspected cholera by culture or PCR.

Number of Samples Required

• Laboratory confirmation by culture or PCR of the first suspected cases is essential to confirm a cholera outbreak.

• Collect stool specimens from the first suspected cases (5–10 cases) and send them to the laboratory for confirmation.

• Declare the outbreak if V. cholerae O1 or O139 is confirmed by culture or PCR with evidence of locally acquired infection (exclude imported cases). The government can declare the outbreak with advice of the international agencies, local authorities and nongovernmental organizations (NGOs)

• Once the outbreak is declared, there is no need to confirm all suspected cases. The clinical case definition is sufficient to monitor epidemiologic trends.

• Determine antimicrobial susceptibility patterns for the first isolates confirmed by the laboratory at the beginning of the outbreak to provide sufficient information to guide antimicrobial treatment.

• Thereafter, carry out periodic sampling from each affected area for confirmation and antimicrobial susceptibility testing (see section 4 – monitoring the outbreak).

  • If RDTs are available, prioritize RDT-positive samples.

  Remember that clinical management of cases is guided by the degree of dehydration of the patient; clinical management does not require RDT testing or laboratory confirmation.

Collection, Storage and Transport of Samples for Confirmation by Culture and PCR

• Accurate and reliable test results depend on having a sample that has been collected, stored, and transported correctly.

• Collect faecal specimens (stool or rectal swabs) from suspected cases within the first 4 days of illness (when pathogens are usually present in highest numbers) and, if possible, before any antimicrobial therapy has been started.

• Do not delay rehydration of patients to take a specimen. Specimens may be collected after rehydration has begun.

• Collect the stool samples and preserve the sample in Cary-Blair transport medium. Cary-Blair transport medium has a shelf life of up to 1 year from date of manufacture and does not require refrigeration (neither before use nor once inoculated). The medium can be used as long as it does not appear dried out, contaminated or discoloured.

• Follow the following steps to preserve samples in Cary-Blair transport medium.

  1. Collect stool samples or rectal swabs

     1. For collection of stool samples:

        • Collect a small amount of stool by inserting a sterile cotton or polyester-tipped swab into the sample and rotating it.

        • Withdraw the swab and examine it to make sure that it carries some visible faecal material.

     2. For collection of rectal swabs

        • Moisten the swab in sterile Cary-Blair transport medium.

        • Insert the swab 2–3 cm beyond the rectal sphincter and rotate.

  2. Immediately place the swab in the transport medium, pushing it to the bottom of the tube.

  3. Break off and discard the top portion of the stick that extends beyond the tube.

  4. Record the patient’s name or initials, specimen number, type of sample, and date of collection on the outside of the Cary-Blair tube.

  5. Dispatch the sample to reach the laboratory within 7 days; it is not necessary to refrigerate the sample.

     • If Cary-Blair transport medium is not available:

       • Collect a fresh stool sample that has not been in contact with chlorine or other disinfecting agent. Place the specimen in a clean container (with no chlorine or any disinfecting agent residue) with a tight-fitting, leakproof lid.

       • Transport to the laboratory within 2 hours at room temperature.

•  If Cary-Blair transport medium is not available and the specimen will not reach the laboratory within 2 hours, use filter paper to preserve and transport the samples as follows.

  1. For culture: use wet filter paper.

     • Culture can be performed from liquid stool samples on filter paper and kept in a moist environment.

     • Using tweezers, dip a small blotting paper disc into a fresh stool sample that has not been in contact with chlorine or other disinfecting agent.

     • Put the sample in a screw-cap microtube and add two or three drops of normal saline solution to stop the sample from drying out. Close the tube well and store at room temperature.

  2. For PCR: use dry filter papers.

     • Dry filter paper can be used for transport of faecal specimens for specific DNA detection by PCR.

     • Place a drop of liquid stool on a dry filter paper and allow it to air dry.

     • Once dry, place the filter paper in a screw-cap micro tube and store at room temperature. Do not add normal saline to the microtube

• All specimens should be sent to the laboratory, address to the cholera focal person, accompanied by a laboratory request form containing, at minimum, the following information: healthcare centre, cholera treatment unit or centre (CTU/CTC) or hospital, patient name or initials, age, place of residence, date and time of collection, symptoms (or treatment plan), RDT results (if performed) and type of testing requested (culture and/or PCR for cholera).

• For all samples:

  • Maintain specimens at ambient temperature at all times. Do not refrigerate specimens, as refrigeration can greatly decrease the population of V. cholerae.

  • Do not allow specimens to dry (unless sending on dry filter paper for PCR). Add additional drops of normal saline solution if necessary.

  • Transport in a well-marked, leakproof container at ambient temperature.

  • Ensure that each specimen and container is properly identified and accompanied by a laboratory.

  • Prior to sending, ensure that the accepting laboratory has the knowledge and capacity to treat the type of sample (for example, wet filter paper for culture, dry filter paper for PCR).

Reporting of Laboratory Results

• Laboratories should maintain an updated database with all samples received and tested and the results of testing.

• Laboratories should send results immediately after completion of testing to:

  • the health facility where the patient was admitted, with identifying information where available so the results can be added to the register and clinic records; and

  • the surveillance unit at the respective health authorities (district, provincial and national level) to update the epidemiological information and the situation report

• Report results of antimicrobial susceptibility testing immediately to the Ministry of Health Clinical Care Services and the health facility to assure appropriate antimicrobial treatment.