Cholera

by J. Nile Barnes

Cholera is a bacterial infection caused by the organism Vibrio cholerae. The bacterium is characterized by short slightly curved rods, which are highly motile (singular polar flagellum), and gram-negative staining. They adhere to the intestinal epithelium and produce enzymes that facilitate access of the bacterium to the epithelial surface. The actual disease symptoms are caused by an exotoxin produced by the bacteria. This exotoxin is commonly known as cholera toxin (CT). (1,2)

Vibrio cholerae is a very common organism. According to Todar (1,2), the natural reservoir for V. cholerae is unknown. It had been thought to have only a human reservoir, but now there is some question as to whether it has a natural aquatic reservoir like many other Virbrio spp. The usual transmission to humans is through contaminated water supplies or food washed or prepared with contaminated water. (1,2)

Robert Koch isolated the bacterium in 1883. Koch's postulates on disease transmission proved Vibrio cholerae was the causative agent in cholera. (3)

Clinical Course

The primary clinical sign of the disease is the sudden onset of massive diarrhea. Patients loose incredible volumes of fluid--up to 10-15 liters/day. The diarrhea is often referred to as "rice-water stools" because of the small flecks of mucous and epithelial cells that are visible in the stool. This dehydration results in significant hypovolemia with its attendant hypotension and hypoperfusion (shock). In addition, severe electrolyte disturbances (primarily hypokalemia) and metabolic acidosis may lead to cardiac dysrythmias and other complaints. Obviously cholera is a rapidly advancing disease. If untreated some patients will die within 2-3 days, however, if a large inoculum is ingested, a healthy person could die within 2-3 hours of the onset of symptoms if untreated. (1,2,4)

Not all infected patients will develop the disease. Not all strains of V. cholerae produce the disease, only two serotypes, designated O1 and O139. In addition, the CDC reports that 75% or more of initial infections with V. cholerae O1 or O139 may not present with symptoms. Of the 25% who do present with illness, most have moderate symptoms and only 2% progress to "cholera gravis" or life-threatening signs and symptoms. There also seems to be a correlation with blood type O. People with blood type O are reported to have a more severe course than those of other blood types. (4)

Mechanism

The disease causing aspect of Vibrio cholera is the secreted exotoxin, (aka cholera toxin). Sometimes this exotoxin is referred to as an enterotoxin. The prefix entero- is of Latin origin and refers to the intestines; an apt name of a toxin whose destructive actions occur in the intestines. This should not be confused with an endotoxin. Endotoxins are not secreted, but are released upon cell lysis. (2)

The diarrhea results from the activity of the cholera toxin which activates adenylate cyclase in the intestinal epithelium, causing them to pump water, Na+, K+, Cl_1, and HCO3-1 from blood and tissues into the lumen of the intestine. The removal of K+ results in hypokalemia and the removal of HCO3-1 causes the metabolic acidosis. (2)

As mentioned above, only V. cholerae O1 and O139 produce the disease. There are actually more than 150 serotypes known. Of the two virulent serotypes O1 is more common than O139 and should be tested for first. Serotype O1 is further divided into two biotypes (genotypes), classical and El Tor. The CDC reports that virtually all worldwide cholera infections are of the El Tor biotype and the classical biotype is not found outside of Bangladesh. In addition to the two biotypes, serotype O1 can be divided into two subserotypes, Inaba and Ogawa. The CDC suggests that documenting the serotype/ subserotype is a worthwhile endeavor, but that it is not necessary to treat an outbreak. (4)

Not all O1 and O139 serotype bacteria are disease causing. There are two genes (two proteins) that are needed, obviously the cholera toxin gene, in addition, a gene to code for a pilus to attach to the epithelial lining of the intestine is required. This pilus is called the toxin-coregulated pilus or TCP pilus. (4)

Microbial Virulence

In the Mekalanos laboratory at Harvard Medical School, researchers have postulated that the presence of cholera toxin (CT) is the result of a bacteriophage infecting the Vibrio cholerae bacteria. This phage is known as the CTX phage. The CTX phage may be inserting the genetic code for the CT protein production. The gene, known as ctxAB, is found in disease causing strains of V. cholerae and the CTX phage but not in isolates that do not cause the disease. This gene is regulated by two genes known as ToxR and ToxT; these are also the genes that regulate the TCP pilus. In addition, the CTX phage is thought to only infect bacteria that have the TCP gene. Therefore, V. cholerae that acquire the ctxAB gene through this mechanism will always produce both the TCP pilus and CT; that is, they will always be virulent. (5,6)

Treatment

Treatment for cholera is aimed at rehydration of the patient. In the US, rapid intravenous isotonic fluid replacement is usually the first line treatment. It is followed by oral hydration using isotonic (or near isotonic) oral hydrationñproducts like GatoradeÆ, PedialyteÆ, and other balanced electrolyte and glucose solutions. In third world countries where sterile intravenous solutions are not as readily available, oral hydration alone is used with considerable success. Antibiotics are considered of little value, in this rapidly advancing disease; by the time the antibiotics are given, the exotoxin is already released and killing the offending organism will not change the toxin levels. Antibiotic (especially tetracycline) use can shorten the length of the illness, but will not affect the severity of the illness. (1,2)

Antibiotics used in cholera (1)

* tetracycline
* doxycycline
* furazolindone
*  trimethoprim-sulfamethoxzole
* erthyromycin
* chloramphenicol
*  ciprofloxacin
* norfloxacin 

Identification

As stated above identification of V. cholerae is not necessary for treatment to begin. Treatment should begin based on the empiric finding of massive diarrhea. In the simplest laboratory test, stool samples can be directly observed by dark-field microscopy for vibiros, but this is not a common method employed for enteric infections. The cholera toxin can be detected using ELIZA (enzyme-linked immunosorbant assay) or latex agglutination testing, however, Forbes, et al, reports that this is not commonly done in the US except for epidemiologic purposes. (7)

Stool cultures for Vibro spp. are done on thiosulfate citrate bile salts sucrose (TCBS) agar; a selective medium. An enrichment broth of alkaline peptone water (pH 8.4) can be used to promote growth. The plate is generally incubated for five to eight hours at 35oC before being subcultured on another TABS plate. Vibrio cholerae colonies will have a yellow color on TCBS. (7)

In terms of biochemical testing V. cholerae will be positive for oxidase, sucrose fermentation, lysine decarboxylase and ornithine decarboxylase. V. cholerae will be negative for d-glucose fermentation, lactose fermentation, myo-inositol fermentation and arginine dihydrolase.(7)

Vibrio cholerae and V. mimicus are the only vibrios that do not require salt for growth; this means a key test to identify V. cholerae from most other vibrios is growth in a 0% salt solution while the others will grow in a 6% solution. (7)

The string test uses 0.5% sodium deoxycholate which lyses vibrio cell walls; it can be used to differentiate vibrios from Aeromonas spp and P. shigelloides. The cell lysis releases the DNA which is "sticky" and can be pulled into a string with a inoculation loop. (7)

Immunity and Vaccination

After a natural infection, "vibriocidal" antibodies can be found to the cholera toxin, flagellar (H) antigens and somatic (O) antigens. The flagellar and somatic antibodies can be induced by injection of the antigens as vaccine componets. The reason they are called "vibriocidal" is that they will lyse vibrios in the presence of complement and sera. These antibodies peak in about a week and return to baseline slowly over a period of two to seven months. After a natural infection, antibodies to the CT are also formed, but there is no correlation between the incidence of cholera and presence of these anti-toxin antibodies. (2)

Immunoglobins IgA, IgG and IgM can all be found in immune persons. The secretory IgA is found in the intestinal mucosa and the IgG and IgM in the serum. Of interest is that the vibrios are found in the intestines where complement is absent. This begs the question "how do the antibodies work?" It is theorized that the anti-H Ab interfere with motility and the anti-O Ab cause aggregation that prevents adherence to the intestinal lining. The anti-toxin Ab could block binding of the toxin to the adenylate cyclase components it activates.  (2)

Because natural infection provides a reasonably lasting immunity (up to approximately one year), there have been efforts to develop vaccines. Past attempts included whole cell preparations and purified lipopolysaccharide fractions of various biotypes. Using formalin and glutaraldehyde the cholera toxin can be converted into a toxioid, but this toxoid has little antigenic properties and offers little immunity. (2)

Todar reports that "attempts are underway to develop an oral vaccine from a live attenuated strain of V.cholerae." The plan is to have the attenuated strain produce a inactive CT molecule that will stimulate immunity but not activate adenylate cyclase. (2)

There is a vaccine to the Bengal strain of serotype O139. Unfortunately, most other cholera outbreaks are due to a O1 serotype, so this vaccine is of limited usefulness and is currently only useful in India and southeast Asia. (2)

In general, the cholera vaccines have proven to be of limited usefulness. In fact, the CDC and WHO no longer recommend travelers going to or coming from endemic areas receive the vaccine. The ACIP (Advisory Committee on Immunization Practices) does note that some countries ("with the threat or occurrence of epidemic cholera") do require a Certificate of Vaccination to enter the country. A Certificate of Vaccination for Cholera is valid from six days after the immunization to six months past the immunization. Revaccination is required for the certificate to be re-validated after six months. (8)

Epidemiology

Several authors have described the endemic cholera of the world as "smoldering." Arno Karlen in Man and Microbes (9) points out that cholera reached Europe as late as the 18th century, but that Sanskrit writings of 2,500 years ago describe this disease in the Bengal region of the Indian subcontient. Others report that Hippocrates describe what may have been cholera in the 4th century BCE (2). Forbes, etal, reports that there have been seven cholera pandemics since 1817 (7). Clearly, this is a global problem.

Cholera has two unique facets that make it interesting to study. First, its mechanism; its seems that its mechanism is the model for exotoxin induced disease (2). This means that nearly every student of microbiology is exposed to this disease concept. The second is that is was the first disease that epidemiology was successfully used to combat. (10)

John Snow was an English physician (anesthesiologist) and some say the first epidemiologist. He is immortalized in epidemiology for making the connection between contaminated water at the Broad Street (see map) pump in 1854 (in London) about which he writes

"Within two hundred and fifty yards of the spot where Cambridge Street joins Broad Street, there were upwards of five hundred fatal attacks of cholera in ten days."

Dr. Snow had already discovered the connection in several cases prior to the Broad Street epidemic (he writes about them in ), but in this case he convinced the authorities (Board of Guardians of St. James's parish) to remove the handle from the water pump on Broad Street--within a week the epidemic ended. (10,11)

The last pandemic seen (the seventh since 1817) lasted ten years, 1961-1971. The spread can be seen on the accompanying map (from Todar (2)).

The 1982 epidemic in Bangladesh is important since is of the classical biotype and not the more common El Tor biotype. No other country has experienced an epidemic of this strain. (2)

Ten years later another epidemic began in Bangladesh, this time of the O139 Bengal variety. Todar reports that it is derived from the El Tor strain but changes in antigenic structure meant no immunity for those with previous exposure to El Tor. (2)

The only current outbreaks are in Southern Africa. In Zambia, as of January 24, 2001 the death toll was up to 22 persons according to the Times of India Online newspaper. The allAfrica.com news site reports that there have been 70 deaths along the Western cape of South Africa since August of 2000. Last April there were over 400 deaths due to cholera reported by the Times of India Online in Southern Africa.  (12,13,14)

Dr. Rita Colwell and Dr. Anwar Huq of the University of Maryland Biotechnology Institute are working on a project called Remote Sensing of Cholera Outbreaks as part of CHAART (The Center for Health Applications of Aerospace Related Technologies). This project is attempting to correlate changes in zooplankton growth in the oceans and seas to cholera outbreaks. This may bring some interesting new ways to map and find isolated outbreaks. (15)

References

1. Todar, Kenneth, Bacteriology 330 Lecture Topics: Cholera (Vibrio cholerae), http://www.bact.wisc.edu/Bact330/lecturecholera, posted 1999, accessed 24 March 2001.

2. Todar, Kenneth, Microbiology Online Cholera, http://www.bact.wisc.edu/MicrotextBook/disease/cholera.html posted 2000, accessed 24 March 2001

3. Paznokas, John, Microbiology 310 Koch's Postulates, http://www.sci.wsu.edu/bio/micro310koch.html posted date not listed, accessed 24 March 2001.

4. Boop, Reis, Wells, Labortory Methods for the Diagnosis of Epidemic Dysentery and Cholera http://www.cdc.gov/ncidod/dbmd/diseaseinfo/cholera/complete.pdf posted 1999, accessed 24 March 2001.

5. GDN Pearson, A Woods, SL Chiang, and JJ Mekalanos ,CTX Genetic Element Encodes a Site-Specific Recombination System and an Intestinal Colonization Factor PNAS 90: 3750-3754. http://www.pnas.org/cgi/reprint/90/8/3750.pdf published April 1993, accessed 29 March 2001.

6. Chiang, Su L., Mekalanos, John J. rfb Mutations in Vibrio cholerae Do Not Affect Surface Production of Toxin-Coregulated Pili but Still Inhibit Intestinal Colonization Infect. Immun. 1999 67: 976-980 http://iai.asm.org/cgi/content/full/67/2/976?view=full&pmid=9916119 published February 1999, accessed 29 March 2001.

7. Forbes, et al, Diagnostic Microbiology, Chapter 35 Vibro, Aeromonas, Plesiomonas shigelloides and Chromobacterium violaceum, pp 488-500, Mosby, 1998.

8. CDC staff, Recommendations of the Immunization Practices Advisory Committee Cholera Vaccine MMWR 37(40);617-618,623-624  http://wonder.cdc.gov/wonder/prevguid/m0042345/m0042345.asp published 14 October 1988, accessed 29 March 2001.

9. Karlen, Arno, Man and Microbes, Touchstone Publishing, 1995.

10. UCLA Department of Epidemiology, John Snow - a historical giant in epidemiology http://www.ph.ucla.edu/epi/snow.html posted date not listed, accessed 24 March 2001

11. John Snow, M.D., On the Mode of Communication of Cholera, London: John Churchill, New Burlington Street, England, 1855 http://www.ph.ucla.edu/epi/snow/snowbook.html published 1855, posted date not listed, accessed 24 March 2001.

12. Staff, Zambia's cholera death toll up to 22 Times of India Online, http://www.timesofindia.com/today/24afrc2.htm posted 24 January 2001, accessed 29 March 2001.

13. Gophe,Myolisi, Western Cape Goes On Full Cholera Alert http://allafrica.com/stories/200101230301.htm posted 23 January 2001, accessed 29 March 2001

14. Staff, SOUTHERN AFRICA: Cholera-hit Countries To Formulate Regional Strategy allAfrica.com,  http://allafrica.com/stories/200101190172.html posted 19 January 2001, accessed 29 March 2001

15. Colwell and Huq, Remote Sensing of Cholera Outbreaks, http://geo.arc.nasa.gov/sge/health/projects/cholera/cholera.html posted date not listed, accessed 29 March 2001