Section 2: Outbreak Confirmation | Cholera Outbreak Response Field Manual

Definitions

Confirmed cholera case

A confirmed cholera case is any person infected with Vibrio cholerae O1 or O139, as confirmed by culture (including seroagglutination) or PCR.
The bacterial strain should also be demonstrated as toxigenic (by PCR) if there is no confirmed cholera outbreak in other surveillance units, and no established epidemiological link to a confirmed cholera case or source of exposure in another country.

Confirmed cholera outbreak

A confirmed cholera outbreak is detected when a surveillance unit has at least one locally acquired, confirmed cholera case.

Laboratory 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 bacterial strain should also be demonstrated as toxigenic (by PCR) if there is no confirmed cholera outbreak in other surveillance units, and no established epidemiological link to a confirmed cholera case or source of exposure in another country.
Conduct antimicrobial susceptibility testing on the index confirmed (i.e., first confirmed) case at a minimum to guide antimicrobial treatment.
Conducting Whole Genome Sequencing on confirmed cholera cases of uncertain origin (i.e., imported cases) is also recommended. However, this is not required for public health intervention.

Collection, conditioning, storage and transport of samples for confirmation by culture and PCR

Accurate and reliable test results rely on samples that have been adequately collected, packed, transported, and stored.
Testing for cholera is performed on patients’ stool.
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.
However, if antimicrobial therapy has been initiated prior to sample collection, information regarding the prescribed antibiotic, dosage, and duration of treatment should be clearly documented in the request form for laboratory testing. Antibiotic therapy may negatively impact laboratory results.
Do not delay rehydration of patients to take a specimen. Specimens may be collected after rehydration protocols have been initiated.

Specimen collection

Able patients: Provide the patient with a container such as a bucket/bedpan without traces of detergent or disinfectant or a new plastic bag (like zip-lock bag) or ideally a wide biodegradable paper cup with a wide enough opening. Instruct the patient to: 1) urinate before using the container and 2) pass stool into the container. The loose stool collected can then be transferred into a stool cup or onto a swab.
Patients who cannot move: Use a clean, unused bedpan or bucket. A bedpan or bucket must be washed, bleached, thoroughly rinsed and well dried before being reused. The bedpan or bucket must not have any residue of chlorine or any other disinfectant. Place it under the hole of a cholera bed or under the patient. Collect freshly passed stool. The loose stool collected can then be transferred into a stool cup or onto a swab.
Rectal swab: In rare instances, a rectal swab may need to be performed. Ask the patient to lie on his side and to bend and lift his knee that is most on top. Moisten the swab in sterile transport medium or with saline. Insert the swab through the rectal sphincter 3–4 cm. Rotate for 5 to 10 seconds, and withdraw with care. Examine to ensure there is faecal material visible on the swab.

Specimen conditioning for transport

There are 5 commonly recommended methods to condition cholera samples for transport to the laboratory.
The selection of the method will depend on what resources are available (stool cups, Cary Blair swabs, etc.), when the sample is expected to be tested (within 2 hours, >2 hours, longer) and what type of tests are to be performed by the laboratory (RDT, culture, PCR etc..). The selection of the method should be made in conjunction with the laboratory.
All samples should be kept and transported at ambient temperature (22 °C – 25 °C) and kept out of direct sunlight.
Fresh sample in a stool cup
- If a sample can reach the laboratory within less than 2 hours, then it is possible to send it directly in the stool cup.
Cary-Blair swab
- If transport to the laboratory may take up to 7 days, it is important to preserve the sample in Cary-Blair medium. Cary-Blair transport medium has a shelf life of up to 1 year from the 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.
- Dip a sterile cotton or polyester-tipped swab into a fresh stool sample and immediately place the swab in a tube of Cary-Blair transport medium, pushing it to the bottom of the tube. If a rectal swab was performed, place the rectal swab directly in a tube of Cary-Blair transport medium, pushing it to the bottom of the tube.
- Break off and discard the top portion of the stick that extends beyond the tube.
Sample in Alkaline Peptone Water (APW)
- Alkaline Peptone Water (APW) is not a transport medium but a medium that favors growth of Vibrio cholerae bacteria. This method should only be used if the sample can reach the laboratory within less than 24 hours.
- Tubes containing sterile APW must be prepared by the laboratory in advance. Transfer fresh stool from the initial container into the tube of APW in a way that the faecal matter should not exceed 10 % of the total volume of the APW.
Sample on wet filter paper
- If Cary-Blair transport medium is not available and the specimen will not reach the laboratory within 2 hours, culture can be performed from liquid stool samples on filter paper and kept in a moist environment (i.e wet filter paper). Samples prepared this way can be tested within 15 days.
- Using tweezers, dip a small filter 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.
Sample on dry filter paper
- Dry filter paper can be used for transport of faecal specimens for specific DNA detection by PCR only. These samples can be stored for a long time.
- Place a drop of liquid stool on dry filter paper and allow it to air dry. Once dry, use tweezers to place the filter paper in a screwcap micro tube or place in an individual pouch and store at room temperature. Do not add saline.
- All specimens should be sent to the laboratory, addressed to the cholera focal person, accompanied by a laboratory request/referral 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 request/referral form.
- 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).

Specimen transport

Triple packaging is required to transport samples collected from cholera suspected cases.
Ensure adherence to national and/or international regulations and train transport services adequately.
Ensure that there are readily available standardised procedures for transport of cholera samples and these take into account local conditions (type of transport, duration of transport).
Samples and sample request/referral forms should be transported together.
For the best quality of stool samples for cholera testing, stool should be kept at temperature ranging between 22 °C and 25 °C. No ice packs are needed unless temperature is expected to go above 35 °C. If ice packs are required, they should be placed between the secondary and tertiary containers and not in direct contact with the stool samples.

Reporting of Laboratory Results

Laboratories should maintain an updated database with all samples received and tested and the results of all testing performed.
Ensure that there are national standardised laboratory reporting procedures and reporting forms with built in quality control measures to ensure minimum levels of reporting and accuracy of reporting.
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 health authorities to update the epidemiological information.
Report results of antimicrobial susceptibility testing immediately to the Ministry of Health Clinical Care Services and the health facility to ensure appropriate antimicrobial treatment.

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