Cholera Workup: Approach Considerations, Stool Examination, Stool Culture

Definitive diagnosis is not a prerequisite for the treatment of patients with cholera. The priority in management of any watery diarrhea is replacing the lost fluid and electrolytes and providing an antimicrobial agent when indicated.

According to World Health Organization (WHO) standard case definition, a case of cholera is suspected when the following conditions are met:

• In an area where the disease is not known to be present, a patient aged 5 years or older develops severe dehydration or dies from acute watery diarrhea

• In an area with a noted cholera epidemic, a patient aged 5 years or older develops acute watery diarrhea, with or without vomiting

In endemic areas, biochemical confirmation and characterization of the isolate are usually unnecessary. However, these tasks may be worthwhile in areas where Vibrio cholerae is an uncommon isolate. If identification of the organism is required, direct microscopic examination of stool (including dark-field examination) is indicated, along with Gram stain, culture, and serotype and biotype identification.

Polymerase chain reaction (PCR) tests for identifying V cholerae have been developed. These have a high degree of sensitivity and specificity. At present, however, such tests are used for screening of food samples.

Vibrio cholerae is a gram-negative curved bacillus that is motile by means of a single flagellum. Laboratory diagnosis is required not only for identification but also for epidemiological purposes (see the image below).

Although observed as a gram-negative organism, the characteristic motility of Vibrio species cannot be identified on a Gram stain, but it is easily seen on direct dark-field examination of the stool.

V cholerae is not fastidious in nutritional requirements for growth. However, it does need an adequate buffering system if fermentable carbohydrate is present because viability is severely compromised if the pH is less than 6, often resulting in autosterilization of the culture. Many of the selective media used to differentiate enteric pathogens do not support the growth of V cholerae.

Routine differential media

Colonies are lactose-negative, like all other intestinal pathogens, but sucrose-positive. When plated onto triple-sugar iron agar to screen for Salmonella and Shigella species, the organism gives the nonpathogenic pattern of an acid (yellow) slant and acid butt because of fermentation of the sucrose contained in triple-sugar iron agar.

Unlike other Enterobacteriaceae, V cholerae is oxidase-positive; hence, in countries where selective media are not available and cholera is not endemic, V cholerae should be suspected if any motile, oxidase-positive, gram-negative rod isolated on routine differential media from the stool of a patient with diarrhea produces an acid reaction on triple sugar iron agar.

Alkaline enrichment media

As Vibrio has the ability to grow at a high pH or in bile salts, which inhibit many other Enterobacteriaceae, peptone water (pH 8.5-9) or selective media containing bile salts (eg, thiosulfate-citrate-bile-sucrose-agar [pH 8.6]) are recommended to facilitate isolation and lab diagnosis. On thiosulfate-citrate-bile-sucrose-agar, the sucrose-fermenting V cholera grow as large, smooth, round yellow colonies that stand out against the blue-green agar.

Specific antisera can be used in immobilization tests. A positive immobilization test result (ie, cessation of motility of the organism) is produced only if the antiserum is specific for the Vibrio type present; the second antiserum serves as a negative control. Vibrio antisera may be unavailable in countries where cholera is not endemic. In endemic regions, this is an excellent quick method of identification, even in small laboratories.

Classic and El Tor biotypes also can be identified using the same method. This is useful for epidemiologic studies.

The major hematologic derangements in patients with cholera derive from the alterations in intravascular volume and electrolyte concentrations.

Hematocrit, serum-specific gravity, and serum protein are elevated in dehydrated patients because of resulting hemoconcentration. When patients are first observed, they generally have a leukocytosis without a left shift.

Serum sodium is usually 130-135 mmol/L, reflecting the substantial loss of sodium in the stool.

Serum potassium usually is normal in the acute phase of the illness, reflecting the exchange of intracellular potassium for extracellular hydrogen ion in an effort to correct the acidosis.

Hyperglycemia may be present, secondary to systemic release of epinephrine, glucagon, and cortisol due to hypovolemia.

Patients have elevated blood urea nitrogen and creatinine levels consistent with prerenal azotemia. The extent of elevation depends on the degree and duration of dehydration.

A reduced bicarbonate level (< 15 mmol/L) and an elevated anion gap occur as a result of increases in serum lactate, protein, and phosphate levels. The arterial pH is usually low (approximately 7.2). Calcium and magnesium levels are usually high as a result of hemoconcentration.