How is Cholera Diagnosed?

How is Cholera Diagnosed?

Cholera is an acute bacteria disease caused by a bacterium called Vibrio cholerae. Infection with the cholera bacteria can lead to severe vomiting and diarrhea and finally electrolyte loss or dehydration. If not treated, cholera can, in many cases, result in death.

In order to treat cholera, there is a need to first diagnose it. Cholera is an infection that is mostly characterized by severe diarrhea, among other symptoms. However, cholera cannot be diagnosed by its symptoms alone since there are many infections with the same symptoms.

Understanding the Cause of Cholera

Cholera is caused by a bacterium scientifically known as Vibrio cholerae. Vibrio cholerae bacterium has further been divided into 130 serogroups based on the presence or absence of somatic O antigens. However, only serogroup O1 is linked to pandemic and epidemic cholera. Other Vibrio cholerae subgroups are only associated with severe diarrhea but do not cause cholera infection. Serogroup O1 is further divided into three serotypes, namely, Ogawa, Inaba and Ikojima.

The identification of the presence of Vibrio cholerae bacteria strains that cause cholera calls for laboratory tests. Laboratory tests involve certain procedures as discussed below:

     1. Symptoms Assessment

The first procedure in diagnosing cholera involves observation of the infection's signs and symptoms. These include:

• Watery diarrhea
• Nausea
• Vomiting
• Abdominal cramps

The above symptoms may leave the patient dehydrated and listless.

    2. Serological Identification

Serological identification is the major laboratory diagnostic test for the Vibrio cholerae bacterium. This procedure involves the use of antisera to identify the serogroup O1 and its serotypes. The isolates of O1 Vibrio cholerae serogroup agglutinate with O1 antisera. This process is used as a confirmation test for this serogroup since other subgroups do not agglutinate with the antisera.

    3. Biochemical identification

Although use of serological analysis can be enough to confirm the presence of Vibrio cholerae bacteria, biochemical confirmation is also important if the agglutination test does not give enough information. The use of TSI, KIA, arginine, lysine, and string test biochemical reactions can help in the screening of fecal and environmental isolates that resemble Vibrio cholerae.

• Oxidase Test. This test involves growing the bacterial strains suspected to be Vibrio cholerae in a non-carbohydrate medium or HIA slant. 2-3 drops of oxidase reagent are applied on filter paper placed in a petri dish. The bacteria culture is then smeared across the wet piece of filter paper using a toothpick. A positive reaction is confirmed by the appearance of a dark purple color after bacterial growth within 10 minutes.
• String Test. This test can be performed on a plastic petri dish or a glass microscope slide. The procedure involves growth for 18-24 hours of bacterial isolates in an HIA medium mixed with 0.5% of sodium deoxycholate aqueous solution. A positive reaction is confirmed if the bacterial cells are lysed by the aqueous solution of sodium deoxycholate. The suspension also loses its turbidity and becomes viscous due to the DNA produced by the lysed cells
• Kligler’s Iron Agar or Triple Sugar Iron Agar Test (KIA or TSI). These tests involve using  carbohydrate-containing media to identify the presence of Vibrio cholerae bacteria. The two tests are very similar but the amount and type of carbohydrates used vary. In the KIA test using glucose and lactose, Vibrio cholerae reacts forming no H₂S gas. The TSI test, which contains glucose and sucrose, reacts in the same manner as the KIA test, giving results of no gas, and no H₂S.

In the KIA and TSI tests, the bacteria are inoculated into the medium surface through the streaking method and then incubated at 35–37 degrees Celsius. They left to stand with loosely-screwed caps and monitored after 18-24 hours.

• Salt Broths. The bacteria culture is inoculated into a salt medium and then incubated at 35-37 degrees Celsius. The inoculates should be very light to make sure no turbidity occurs before the broths are incubated. The latter are observed after 18-24 hours.
• Voges Proskauer Test. The bacterium or organism under test is inoculated and then incubated in a MR-VP broth for about 48 hours before the addition of 1% sodium chloride, naphthol in absolute ethanol (solution A), and 0.3% creatine dissolved in 40% potassium hydroxide (solution B). The sensitivity of this test is increased by the addition of solutions A and B. The positive result of this test is the formation of a cherry red color.
• Arginine Glucose and Lysine Iron Agar Tests. The lysine iron agar and arginine glucose slant are tests used to test for the production of lysine decarboxylase and arginine dihydrolase respectively. The organisms in the test are inoculated into the LIA or AGS media through the streaking method and then left to stand for 24 hours. A positive reaction in the LIA or AGS media is the production of an alkaline forming a purple color. A negative reaction involves the formation of an alkaline purple color with an acid butt of yellow color.
• Carbohydrates. This involves inoculation of freshly-grown bacteria into sucrose and glucose broths. The broths are then incubated at 35-37 degrees Celsius and then read after 24 hours. A positive test is indicated by the production of a pink color on the Andrade indicator due to acid production. 

     4.  Hemolytic Test

In this test, blood agar plates filled with 5-10% sheep's blood are streaked to get the bacterial colonies. The agar plates are then incubated at 35-37 degrees Celsius and observed after 18- 24 hours. The haemolysed colonies are expected to have clear zones at points where the erythrocytes are fully lysed. The suspected strain of bacteria which has been haemolysed as well is compared with a control strain which has been strongly haemolysed. Non-hemolytic Vibrio cholerae bacteria produce a greenish color on aerobic sheep's blood agar and this reaction acts as a confirmation test.

     5. Dipstick Strip Test.

This test is mostly used in cholera-endemic areas. It is commonly referred to as the immunochromatographic dipstick testing. This test mostly involves inserting a dipstick into a sample of human stool and then reading the lines it displays. Vibrio cholerae bacteria is confirmed in stool if 2 red lines appear on the dipstick. If only one red line appears on the dipstick, the test is negative. It takes about 2-15 minutes for the lines to appear.

Bottom Line

Diagnosing cholera is very important before its treatment. This will avoid treating the infection merely on the basis of symptoms, which may be caused other infections. Although signs and symptoms are considered the first-line diagnosis before other tests are performed, they cannot provide confirmation. This calls for other lab tests as summarized below:

• Dipstick strip test
• Hemolysis test
• Biochemical identification
• Serological identification

The above tests are different from each other as they use different samples and different reagents.