What are the best detection medias for cholera?

What are the best detection medias for cholera?

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I heard this fact that you can use some [hypertriade] for vibrio cholera diagnosis which has compontents

  • sucrose (yellow)
  • mannose (yellow)
  • arabinose (do not ferment; stay dark pink)

I did not find this hypertriade agar on Google. It reminds me of Egg Yolk Agar, but I think cannot be it because of the arabinose. Otherwise, it seems to be similar.

What are the best detection medias for cholera?

You should use Thiosulfate-citrate-bile salts-sucrose agar for the detection of all vibros, including cholera. When cholera is presant, you should expect round, largish (not huge), yellow colonies. This is the CDC recommended diagnoses method.

Again this question might be better phrased "what are the best detection medias for cholera," or "what plate screens should be conducted when vibros are suspected?"

Cholera is an extremely virulent disease that can cause severe acute watery diarrhoea. It takes between 12 hours and 5 days for a person to show symptoms after ingesting contaminated food or water (2). Cholera affects both children and adults and can kill within hours if untreated.

Most people infected with V. cholerae do not develop any symptoms, although the bacteria are present in their faeces for 1-10 days after infection and are shed back into the environment, potentially infecting other people.

Among people who develop symptoms, the majority have mild or moderate symptoms, while a minority develop acute watery diarrhoea with severe dehydration. This can lead to death if left untreated.


Cholera is a waterborne disease [1–2] which leads to a life-threatening acute watery diarrhea [2–5]. In 2017 over 1.2 million cases of cholera resulting in over 5600 deaths have been reported to the World Health Organization (WHO) [3]. It is estimated that only 5–10% of the cases and deaths are reported, which leads to the estimation (from 2014) that there are between 1.3 and 4 million cases each year, leading to some 21 to 143 thousand deaths/year worldwide [6]. The number of reported cases and deaths in 2017 was much higher than at the time the total numbers were estimated (2014), due to an outbreak in Yemen which was responsible for 84% of the WHO’s reported cases and 41% of the deaths alone [3]. As cholera is mainly affecting the poor [7] and is often spread in suburban areas, it is important to have a cheap on-site test which is not depending on the availability of well-equipped laboratories. Cholera has a short median incubation period of 1.4 days, and the symptoms are visible within 4.4 days (95%) [4]. As it can become epidemic, quick tests are needed. The WHO’s global task force on cholera control named the development and the availability of such a rapid test as part of one of their 3 axes to fight cholera [7].

Cholera is a result of intoxication with Vibrio cholerae (V. cholerae), a bacterium which produces cholera toxin (CT). Around 200 serogroups of V. cholerae are known today, two of which (O1 and 0139) are the most dangerous and can become epidemic [1–2,4]. CT is a heterohexameric toxin, consisting of a single A subunit (CTA) and a homopentameric B subunit (CTB) [8]. With this structure, it belongs to the group of AB5 toxins, which are medically important toxins that constitute important virulence factors [9–10]. The A subunit of CT is the catalytic active part which leads to the disruption of essential host functions. It can be divided into two sections [2,9]. The first section (CTA1) is responsible for the toxicity by means of an increased generation of cyclic adenosine monophosphate (cAMP) in the cytosol which leads to a chloride ion (Cl-) secretion [2,11]. To keep the osmolality, this results in a water outflow into the intestinal lumen and cell death. This increased water outflow leads to the symptomatic diarrhea and therefore to a reduction of the blood volume [8]. The second section CTA2 is responsible for the noncovalent anchoring of CTA inside the homopentameric CTB. The ring-shaped CTB binds to the monosialotetrahexosylganglioside (GM1) receptors on mammalian intestinal epithelial cells with high affinity. As each monomer of the CTB pentamer has a receptor binding site, it can bind to five GM1 receptors at a time. It has been shown that only one functional binding site is sufficient for the intoxication pathway, but with a reduced activity [11]. The whole CT is then endocytosed into the cell and retrograde transported to the endoplasmic reticulum where CTA1 is separated [9,11,12]. CTB is not toxic, and CTA needs CTB as a transport vehicle inside the cell. Because of this, only the whole CT complex is intoxicating cells, and not one subunit alone.

The detection of CTB is not only relevant for the diagnosis of cholera or drinking water safety analysis but it is currently also used in different other applications. For example, it is used as a vaccine adjuvant or anti-inflammatory agent [2] or as a mucosal immunomodulatory agent [13].

Different techniques exist for the detection of Cholera or CTB, respectively [14–18]. One form is the direct cultivation of V. cholerae on a medium which is considered as the gold standard [14]. This method has the advantage that the detection sensitivity can be increased by longer incubation, but that is time consuming (about 24 hours [14,15,17,19,20]), and the amount of health-threatening material is amplified. Additionally, special microbiological equipment and incubators are required, as well as microbiologically skilled personnel. With this approach, only living bacteria can be detected. Different rapid diagnostic tests based on immunochromatographic methods are commercially available and have been compared to the gold standard during different outbreaks of cholera [20]. Another possibility is polymerase chain reaction (PCR) to detect the bacteria’s deoxyribonucleic acid (DNA) [15]. Here it is important that the DNA is not destroyed during sample handling or due to previous environmental conditions. As expensive laboratory equipment and skilled personnel is required, it is not so practicable for in-field use. With this technique, it is not possible to detect the toxin but only the bacterial DNA. As the toxin itself leads to the symptoms, it might be preferable to detect it directly, especially since the efficiency of antibody-based assays recognizing V. cholerae in general are highly dependent on the binding specificity between antibody and antigen. Thus immune-based approaches, which can specifically detect the strains harmful for humans from all different serotypes of V. cholerae, are difficult to establish. As toxins can withstand different environmental conditions than the bacteria and are not affected by antibiotic treatment, this direct testing can be favorable. It should also be mentioned that, for example, in cases of bioterrorism, where the pure toxin is used instead of the whole bacteria, a bacteria/DNA based detection method would not work. The authors in [18] mention that there is an “urgent demand for rapid and accurate determination of bacterial toxins” and give CT as an example. For the direct detection of the toxin, the nontoxic B subunit can be used. As mentioned before, the A subunit alone is not able to intoxicate a patient. Therefore the CTB detection is sufficient. As CTB has a homopentameric structure which binds up to five receptors at a time, it is possible to bring more than one marker to a CTB unit at a time. This possibility of more than one binding site is used, for example, in the latex agglomeration test [15], in colorimetric assays or in dynamic light scattering assays [21]. Another approach is to use fluorescence labels or radioimmunoassays [22]. An often used method is the enzyme-linked immunosorbent assay (ELISA) [15,22]. Superparamagnetic beads (MBs) have been used to capture CTB magnetically and to afterwards measure them with an attached fluorescent dye or electrochemical marker [23]. Here the MBs are used as handles, like in separation or targeting setups [24].

In this research, we decided to directly use superparamagnetic beads as a marker to determine the achievable detection limit and the quality of the calibration curve. In modern bioanalytical and biomedical applications [16,24,25], MBs are widely used as labels, handles, or both. Different labels can be used in immunologic detection technologies, for instance, enzymes, fluorophores, radioisotopes, or magnetic beads [26,27]. In ELISAs, the linked enzymes catalyze a reaction leading to a color change. This behavior as well as the reaction of the fluorophores might be difficult to detect if the sample is colored or turbid. Another disadvantage of markers like radioisotopes or fluorophores is that their signal changes over time because they have a half-life or undergo photo bleaching. In contrast, the signal of the MBs do not change over time and, therefore, can be read out at a later time or read out later again [28]. For the detection of MBs, different magnetic sensors can be used, for example, coils, giant magnetoresistance (GMR) sensors [29], and superconducting quantum interference devices (SQUIDs) [30]. Common techniques for magnetic measurements are, for example, relaxometry [31], susceptometry [32], and nuclear magnetic resonance (NMR) [33]. We used the magnetic frequency mixing technique [34–38] to develop a simple rapid detection system for CTB. In [36–38], this technique has been compared with ELISA measurements and its applicability for immunoassays has been shown. As CTB is in many application cases solved in a liquid-like medium (drinking water, diarrhea stool), a detection method which can directly make use of this is favorable. Therefore we used a sandwich immunoassay inside a three-dimensional (3D) immunofiltration column.

Presumptive diagnosis:

I) Immobilization test : A rapid presumptive diagnosis of cholera can be made by observing the wet smear for the distinctive rapid to and fro movement (darting movement) of V. cholerae O1 and O139 due to their single polar flagellum . The movement can be stopped by adding one drop of V. cholerae O1 and O139 antiserum respectively.

Hanging drop method for Vibrio cholerae is one of the easy but most popular tests used for the presumptive diagnosis. Read details about this test here

II) Oxidase test: on performing an oxidase test from a pure sub-culture on nutrient agar, a positive reaction is observed. Oxidase test should not be performed directly from TCBS or MacConkey agar as acidification of these media may result in false-negative oxidase tests.
(Note: However, Aeromonas spp also gives a positive oxidase test result so further confirmation is necessary by culture and serotyping)


Small intestine colonization is highlighted by V. cholera's highly effective motility andꃪse ofਊttachment to the intestinal wall. V.਌holerae requires a comparatively high infectious dose (10^8).[10]  Cholera toxin is then secreted and eventually endocytosed by the intestinal epithelial cells, altering the electrolyte channels, and resulting in endoluminal fluid loss rich in chloride, bicarbonate, sodium, and potassium.[11] On excretion into the environment, it has been found that the bacteria undergo a period ofꀤ hours of hyper-infectious activity and are more likely to be transmitted in a human-to-human fashion, explaining the explosive nature of Cholera਎pidemics.[12]ਊnother important pathophysiological feature of V. cholera is how host susceptibility affects a patient's risk. For example, individuals with blood group O have been found to be more likely to develop severe Cholera than other blood types,[13][14] while individuals previously infected with Cholera or vaccinated against it have often򠯮n found to gain temporary acquired immunity.[15] Recently, there has been an increase in the number of non-O1 and non-O139 V. cholera infections presenting as self-limited gastroenteritis after bathing in contaminated recreational waters or ingestion of raw and under-cooked seafood.[16][5]

Postbac Spotlight—Translational Research, The Best of Both Worlds

Sarah Cook is a postbac in the Molecular Development of the Immune System Section of the Laboratory of Immune System Biology. Read Sarah’s postbac spotlight, where she details her postbac experience in the unique setting of the NIH Clinical Center and how this experience has influenced her decision to pursue a career in medicine and translational research.

Sarah Cook is a postbac in the Molecular Development of the Immune System Section, Laboratory of Immune System Biology. Sarah details her postbac experience in the unique setting of the NIH Clinical Center and how this experience has influenced her decision to pursue a career in medicine and translational research.

The NIH Clinical Center is a unique place to do research. It’s been especially helpful for me, as someone who was unsure about how to fuse my previous research interests with a growing interest in medicine, to learn about the translational research going on in NIAID. I’ve been working in the Molecular Development of the Immune System Section of the Laboratory of Immune System Biology, which studies patients with a spectrum of rare, inherited immunodeficiencies. These monogenic diseases are actually quite complex and difficult to diagnose because of overlapping clinical symptoms and incomplete trait penetrance. We use whole exome sequencing on patient samples to discover potential causal genetic variants that we can validate functionally with model systems.

Studying the cellular and biochemical effects of variants can offer new insights into mechanisms of the immune system. For example, I’ve been working closely with Dr. William Comrie, my postdoc mentor, to characterize an actin-related disease that was discovered in only five patients. These patients have biallelic loss-of-function mutations in the gene NCKAP1L, which encodes a protein called Hem1 and is expressed specifically in the immune system. We were able to distinguish abnormal cellular functions related to defective actin polymerization—like impaired neutrophil chemotaxis and T-cell immune synapse formation—from those that weren’t expected based on known functions of the protein. In this way, we discovered a novel connection between Hem1 and the mTORC2 regulatory system.

It’s been exciting and challenging to incorporate a molecular understanding of immunology with clinical data from patients. This experience has definitely guided my decision to apply to research-oriented medical schools.

What are the best detection medias for cholera? - Biology


Cholera is one of the most feared clinical entities on earth. Outbreaks in India have been well documented since the early 1800’s, in which hundreds of thousands of people became ill. Many of those who got sick went on to die. The organism responsible for this serious diarrheal disease was most likely present in human populations on that subcontinent well before the British arrived there. One of the first documented epidemics of cholera occurred in 1817 along the coastal region near the mouth of the Ganges River. Cholera now has a worldwide presence, with many people dying each year. Most deaths from cholera can be avoided if adequate medical care were made available.

Cholera spread rapidly throughout the world after the 1817 epidemic, largely due to the inadvertent transport of bilge water, mainly from British ships, but others too, acquired in the Bay of Bengal that contained the organisms. Dumping the contaminated water into their own port cites upon arrival home seeded the local waters with it and insured the eventuality of an outbreak. It then rapidly moved throughout Europe and into Russia. The French were the ones who brought it to the New World, and in 1832, it spread south from Montreal and caused an enormous epidemicin New York City (see: The Cholera Years, by C. E. Rosenberg, The University of Chicago Press, 1987, pp 265). In 1855, a wave of cholera ravaged the citizens of some parts of London. Thousands became ill and died before the medical detective work by John Snow identified the Broad Street water pump as the single point source of that outbreak. His classical maps showing where people who became sick lived convinced him that the only possible source of the infection was the water pump. This landmark study established the epidemiological view of cholera that has endured until quite recently. In London on the corner of Broadwick (formerly Broad) stands the John Snow pub, a fitting commemorative honoring the site on which these historic events unfolded. Today, all patrons of the John Snow can enjoy a pint of local ale, and even more importantly, a refreshing glass of crystal clear, pathogen-free water. Since 1961, there have been seven major cholera pandemics (The global spread of cholera during the seventh pandemic, 1961-1971 [source]), affectingmillions of people living in South America, Africa, Europe, and Asia. To fully appreciate its biology, one must take into account data collected from many different scientific disciplines. Ecology, molecular biology, microbiology, epidemiology, pathology, and long range sensing all have supplied critical pieces of information, which, taken together and integrated, forms a comprehensive body of knowledge as to how cholera enters the human population and what factors regulate its occurrence within the estuary. Thus, cholera is a perfectly suited topic for illustrating the usefulness of the Medical Ecology paradigm.

The Cholera Organism

First isolated, cultured, and characterized by Robert Koch in Germany in 1883, the organism is a comma-shaped, flagellated, gram-negative bacterium, Vibrio cholerae. In fact, it was Koch’s work on cholera the led the way to firmly establishing the germ theory of disease, and helped convince the medical community as to the microbial nature of this devastating clinical condition. For all of his exemplary work, he was the recipient of the Nobel Prize in Medicine in 1905. In the laboratory, it can be easily grown at 37°C on blood agar, as well as on selective media such as thio-citrate-bile salt-sucrose. There are many 16 strains of V. cholerae, and the 01 and 0139 strains are the most lethal. While V. cholerae is the best characterized of these agents, several other species of Vibrio can also cause significant disease.

Pathological strains produce clinical symptoms and signs, the most common one by far being a protracted, watery diarrhea. Yet, despite the fact that human populations are routinely infected with it, V. cholerae’s natural habitat is not our small intestine, since most infections last for only several days, and the carrier state in humans is extremely rare. It was well into the 21st century before its fundamental niche was revealed to be the estuary, a narrow ecological region known as an ecotone. Typically, the first clinical cases of any new outbreak occur in communities situated on or near an estuary. Although this fact was known for at least since the 1800s, it was not considered essential to the natural history of the disease. In fact, its ecological role, once revealed, surprised even those who resolutely suspected that it was essentially an organism that occupied a fundamental niche outside the human host, but could not prove it.

How Do Cholera Epidemics Start?

One of the enigmas related both to its ecology and to human disease was its apparent absence from human populations just prior to epidemics. Besides the fact that extensive clinical research repeatedly failed to identify human carrier states as the source, the bacterium did not form spores, so a resting stage could not be demonstrated in the estuarian environment. V. cholerae seemed to simply “disappear” at times when cases were not occurring (prior to the arrival of the monsoons in South Asia, for example). How does an epidemic get its start if humans are not the source of the initial infection? Could there be reservoir animal species that harbored the organism and occasionally contaminated the human environment, or perhaps there was a stage of the bacterium that was more difficult to find that a spore stage that allowed it to survive in salt water.

Monsoons represent seasonal patterns of precipitation that bring with them changes in both the relative salinity and temperature of the estuaries of major river systems along the entire coast of the Indian subcontinent. These and other related seasonal precipitation events in similar tropical and sub-tropical environments cause dramatic shifts in the aquatic environment, triggering blooms of phytoplankton, that in turn serve as the food source for a rich assortment of zooplankton grazer species. It has been known from the time of Koch that cholera organisms grow best at a temperatures above 17°C, and in a nutrient broth with a [NaCl] of 5-15 parts per million, well below that of the open ocean, but above that of freshwater. Those conditions are met in the estuaries by episodes of heavy rains in the spring, and appear to be absolutely essential to establishing an ecological setting favoring an outbreak. But where do the organisms come from? After years of intensive laboratory and field studies, it was discovered that many species of copepods that comprise the myriad assemblages of zooplankton communities in those estuaries harbor V. cholerae as an ecto-symbiont. Organisms can be found growing on their egg sacks and inside their gut tracts. This discovery opened the way for a more complete description of the ecology of the cholera group of bacteria.

There still, however, remained unanswered questions regarding its epidemic nature. One large missing piece of the puzzle was the fact that zooplankton blooms are not always present in the estuary, and hence cholera cannot routinely be cultured from most brackish water environments. Where did the microorganisms go during quiescent periods in between seasonal rain events? Further laboratory-based research revealed that they could transform into a unique dormant stage that was able to survive for months in the sediment of the estuary. This stage was unlike spores of bacteria such as Bacillus subtilis, an aerobic, non-pathogenic, soil-dwelling organism, or its anaerobic cousin, Clostridium perfringens, a soil-dwelling bacterium capable of causing acute gastroenteritis and gas gangrene. These exciting new data enabled investigators to now integrate information regarding the seasonal nature of events surrounding an outbreak in populations living near estuaries with previous data on the physical and chemical requirements for its growth. What has emerged over the last 10 years has quite literally revolutionized the way we look at cholera.

A Modern Synthesis

Seasonally dependent epidemics can now be described in ecological terms. Warm, intense episodes of precipitation falling on coastal and nearby inland regions transforms the temperature and salinity profiles of estuaries. These changes create favorable growth conditions for the dormant bacterium, and also, most importantly, for phytoplankton species. The influx of large quantities of freshwater mobilizes stored nutrients in the bottom sediments of the estuary, and gives the cholera bacterium a head start in its growth cycle. Algal blooms in response to higher temperatures, lower salinity, and nutrient loading allow for a similar increase in filter feeding zooplankton. Nutrient loading of the estuary typically occurs from a variety of nearby riparian sites (point source and non-point source run off), thus serving as the final environmental cue, supplying V. cholerae with additional sources of nutrients. This apparently is sufficient enough to permit an increase in bacterial cell numbers to a level that enables the organism to encounter copepods of the right species. Copepod species then pick up the bacteria on their external and internal surfaces. This most likely involves specific bacterial ligands and copepod surface receptor molecules, yet to be identified and characterized. Once the cholera organism attaches, the crustaceans carry the bacteria along as a normal component of their bodies throughout their life cycle. The bacterium continues to replicate until they completely cover the surface of the copepod’s egg sack. When eggs mature, the overall process apparently triggers the cholera bacteria to synthesize, then secrete a chitinase that functions to dissolve the outer egg case, facilitating the release of the eggs, dispersing them and the bacteria into the water column. The more dense the population of zooplankton, the more the concentration of free cholera bacteria there will be in the water column. Filter feeding benthic organisms (e.g. crabs, clams, and oysters) process large volumes of water, concentrating particulates and the cholera bacteria in their gut tracts. Humans that harvest these contaminated food organisms in spring-time, and ingest them raw place themselves and the rest of their local communities at risk from acquiring cholera. A bacterial cell concentration of 10 3 /ml of water is necessary to allow an infectious dose of V. cholerae to accumulate within mollusks and crustaceans.

Application of Ecological Knowledge to the Control of Cholera

When a single case of diarrheal disease due to V. cholerae occurs, it has the potential of spreading into the local water supply, and contaminating entire villages, and sometimes whole coastal regions. Remote sensing from a variety of orbiting earth-monitoring satellites can simultaneously detect changes in weather patterns and phytoplankton blooms. Other satellites can determine sea surface temperature changes. These data, taken in combination, after further refinement, could eventually become the lynch pin in a network of data collection whose sole purpose would be to predict the next cholera epidemic. Early warning of coastal inhabitants based on this premise could result in millions of lives spared from the ravages of this age-old human pathogen.

This new view of cholera was championed by Rita Colwell and colleagues. The salient features of its ecology took many years of hard work and insight. The scientific community at-large, bounded by a more traditional (i.e., John Snow/Robert Koch) view of cholera, unofficially encouraged wide spread opposition to many of the then radical hypotheses that, in fact, turned out to be validated in a series of elegantly conducted laboratory and field studies. This attitude resulted in significant delays in getting relevant data into peer-reviewed journals. Unshakable perseverance and a firm belief in the principles of Medical Ecology won the day. Currently, Rita Colwell is the director of the National Science Foundation.

More Cholera Mysteries Solved

Another aspect of cholera that was not understood was why its virulence varied greatly from strain to strain. Some strains even failed to produce disease. Cholera toxin, an enzyme, was eventually identified as the main virulence factor associated with strains that induced acute diarrhea. Cholera toxin is synthesized and secreted by strains in the 01 and 0139 groups, only. Those lacking this enzyme are far less pathogenic. Its mode of action eventually results in prolonged hypersecretion in the small intestine. The diarrhea is so intense that enterocytes become fragile and begin to sluff off from the basement membrane of the villus soon after symptoms appear.

Cholera toxin attaches at the level of the crypts of Lieberkühn to enterocytes that have surface ganglioside Gm1, a special glycolipid. Internalization of the toxin-ganglioside complex then occurs. The bacterial enzyme catalyses the transfer of ADP ribose from intracellular NAD+ to the s subunit of the trimeric G protein that is normally attached to the cytoplasmic side of the plasma membrane of each enterocyte. ADP ribosylation changes the activity of s subunit so it can no longer hydrolyze its bound GTP substrate, thus deregulating cyclic AMP activity. Hypersecretion immediately ensues. Efflux in chloride and bicarbonate ions into the small intestinal lumen pulls large quantities of water with it by passive osmosis. The process continues until no more toxin is produced, or until the enterocyte is shed into the lumen of the small intestine.

The acute condition caused by cholera toxin is known as "rice water" stool, because the free enterocytes in the almost clear liquid stool give the appearance of rice grains. Oral re-hydration with saline solution is the recommended supportive therapy that has saved countless millions of lives. Antibiotics can reduce the length of time for the diarrheal portion of the illness, but is often in short supply or not available at all. The cholera organism "burns out" on its own within 5-6 days and the patient experiences an uneventful recovery.

The cDNA encoding the toxin has been cloned and sequenced, and the putative protein expressed and characterized. Interestingly, that data clearly showed that cholera toxin was not related to any known protein produced by non-pathogenic strains of Vibrio. Rather, the protein showed large regions of homology and similarity to a family of endotoxins produced by bacteria in a completely different family, the Enterobacteriaciae (e.g., Escherichia coli, Shigella spp. and Salmonella spp.). Vibrios are in the family Vibrionaceae.

How did Vibrio acquire its enterobacteriaciae-like toxin? Since not all varieties of the cholera bacteria have the toxin, it was hypothesized that the gene encoding it might be the result of a latent phage infecting only a few of them (01 and 0139). This suspicion was eventually confirmed, and the phage, as well as the DNA coding region for the toxin, has now been fully described for strains 01 and 0139 by Mekalanos [link2] and colleagues.

Yet, despite this new insight into the pathogenicity of Vibrio cholerae, some important questions still remain unanswered. How and when did these two cholera strains acquire the gene for the toxin molecule? Does the toxin offer selective advantage to the two strains in their estuarine niche or in the human host? Can other species of vibrio acquire the gene encoding the toxin? The answers to these questions await further investigation. In 1999, scientists in New Delhi, India described two new strains of vibrio that are resistant to a variety of standard antibiotics and induce severe diarrhea. They are being referred to as enteropathogenic Vibrio cholerae, or EPVC. Neither contains the classical cholera toxin molecule, but they apparently have other virulence factors that produce a diarrhea similar to Shigella sp. and toxigenic Escherichia coli.

Within the last few years, marine scientists have come to realize that the open ocean is teeming with viruses (1-10 million viral particles/ml) of a surprisingly wide range of types. Not all of these viral particles are infectious, as UV radiation inactivates most of them in the photic zone. However, it is likely that at least a small portion of them escape damage long enough to infect numerous species of microbes. In the distant past, within the estuary, similar phages most likely gave V. cholerae 01 and 0139 a version of E. coli’s toxin, which has since that time, evolved somewhat from the parent molecule. The overall process in reminiscent of the mechanisms by which antibiotic resistance can and does occur through associations between bacteria and viruses in the gut tract of humans and farm-raised animals, such as chickens, pigs, and cows.

In the meantime, it seems probable that genomic exchanges between dissimilar organisms in estuaries is commonplace, and can rarely result in the emergence of virulent strains of otherwise harmless microbes. The significance of is concept towards explaining the origins of new infectious agents throughout the world cannot be emphasized enough. In order to become more predictive regarding epidemics sharing similar ecological features to that of cholera, we will need to become even more vigilant of the subtle environmental changes in the world's estuaries induced by an ever increasing human population, or suffer the consequences.

Symptoms and treatment

Cholera is marked by the sudden onset of profuse, watery diarrhea, typically after an incubation period of 12 to 28 hours. The fluid stools, commonly referred to as “rice water” stools, often contain flecks of mucus. The diarrhea is frequently accompanied by vomiting, and the patient rapidly becomes dehydrated. The patient is very thirsty and has a dry tongue. The blood pressure falls, the pulse becomes faint, and muscular cramps may become severe. The patient’s eyes become hollow and sunken, and the skin becomes wrinkled, giving the hands the appearance of “washerwoman’s hands.” Children may also experience fever, lethargy, and seizures as a result of the extreme dehydration. The disease ordinarily runs its course in two to seven days.

The rapid loss of fluid from the bowel can, if untreated, lead to death—sometimes within hours—in more than 50 percent of those stricken. However, with proper modern treatment, mortality can essentially be prevented, with rates kept to less than 1 percent of those requiring therapy. This treatment consists largely of replacing lost fluid and salts with the oral or intravenous administration of an alkaline solution of sodium chloride. For oral rehydration the solution is made by using oral rehydration salts (ORS)—a measured mixture of glucose, sodium chloride, potassium chloride, and trisodium citrate. The mixture can be prepackaged and administered by nonmedical personnel, allowing cholera to be treated even under the most adverse conditions. ORS can generally be used to treat all but the most severely dehydrated patients, who require intravenous rehydration.

The administration of antibiotics such as tetracycline during the first day of treatment usually shortens the period of diarrhea and decreases the amount of fluid replacement required. It is also important for patients to resume eating as soon as they are able in order to avoid malnutrition or to prevent existing malnutrition from becoming worse.

Vibrio Cholerae

Clinical Manifestations

Following an incubation period of 6 to 48 hours, cholera begins with the abrupt onset of watery diarrhea (Fig. 24-1). The initial stool may exceed 1 L, and several liters of fluid may be secreted within hours, leading to hypovolemic shock. Vomiting usually accompanies the diarrheal episodes. Muscle cramps may occur as water and electrolytes are lost from body tissues. Loss of skin turgor, scaphoid abdomen, and weak pulse are characteristic of cholera. Various degrees of fluid and electrolyte loss are observed, including mild and subclinical cases. The disease runs its course in 2 to 7 days the outcome depends upon the extent of water and electrolyte loss and the adequacy of water and electrolyte repletion therapy. Death can occur from hypovolemic shock, metabolic acidosis, and uremia resulting from acute tubular necrosis.

Figure 24-1

Pathophysiology of cholera.

Structure, Classification, and Antigenic Types

The cholera vibrios are Gram-negative, slightly curved rods whose motility depends on a single polar flagellum. Their nutritional requirements are simple. Fresh isolates are prototrophic (i.e., they grow in media containing an inorganic nitrogen source, a utilizable carbohydrate, and appropriate minerals). In adequate media, they grow rapidly with a generation time of less than 30 minutes. Although they reach higher population densities when grown with vigorous aeration, they can also grow anaerobically. Vibrios are sensitive to low pH and die rapidly in solutions below pH 6 however, they are quite tolerant of alkaline conditions. This tolerance has been exploited in the choice of media used for their isolation and diagnosis.

Until 1992, the vibrios that caused epidemic cholera were subdivided into two biotypes: classical and El Tor. Classical V cholerae was first isolated by Koch in 1883. Subsequently, in the early 1900s, some vibrios resembling V cholerae were isolated from Mecca-bound pilgrims at the quarantine station at El Tor, in the Sinai peninsula, that had been established to try to control cholera associated with pilgrimages to Mecca. These vibrios resembled classical V cholerae in many ways but caused lysis of goat or sheep erythrocytes in a test known as the Greig test. Because the pilgrims from whom they were isolated did not have cholera, these hemolytic El Tor vibrios were regarded as relatively insignificant except for the possibility of confusion with true cholera vibrios. In the 1930s, similar hemolytic vibrios were associated with relatively restricted outbreaks of diarrheal disease, called paracholera, in the Celebes. In 1961, cholera caused by El Tor vibrios erupted in Hong Kong and spread virtually worldwide. Although in the course of this pandemic most V cholerae biotype El Tor strains lost their hemolytic activity, a number of ancillary tests differentiate them from vibrios of the classical biotype.

The operational serology of the cholera vibrios which belong in O antigen group 1 is relatively simple. Both biotypes (El Tor and classical) contain two major serotypes, Inaba and Ogawa (Fig. 24-2). These serotypes are differentiated in agglutination and vibriocidal antibody tests on the basis of their dominant heat-stable lipopolysaccharide somatic antigens. The cholera group has a common antigen, A, and the serotypes are differentiated by the type-specific antigens, B (Ogawa) and C (Inaba). An additional serotype, Hikojima, which has both specific antigens, is rare. V cholerae O139 appears to have been derived from the pandemic El Tor biotype but has lost the characteristic O1 somatic antigen it has gained the ability to produce a polysaccharide capsule it produces the same cholera enterotoxin and it seems to have retained the epidemic potential of O1 strains.

Figure 24-2

Vibrio cholerae (O group 1 antigen).

Other antigenic components of the vibrios, such as outer membrane protein antigens, have not been extensively studied. The cholera vibrios also have common flagellar antigens. Cross-reactions with Brucella and Citrobacter species have been reported. Because of DNA relatedness and other similarities, other vibrios formerly called “nonagglutinable” are now classified as V cholerae. The term nonagglutinable is a misnomer because it implies that these vibrios are not agglutinable in fact, they are not agglutinable in antisera against the O antigen group 1 cholera vibrios, but they are agglutinable in their own specific antisera. More than 139 serotypes are now recognized. Some strains of non-O group 1 V cholerae cause diarrheal disease by means of an enterotoxin related to the cholera enterotoxin and, perhaps, by other mechanisms, but these strains have not been associated with devastating outbreaks like those caused by the true cholera vibrios. Recently, vibrio strains that agglutinate in some O group 1 cholera diagnostic antisera but not in others have been isolated from environmental sources. Volunteer feeding experiments have shown that these atypical O group 1 vibrios are not enteropathogenic in humans. Recent studies using specific toxin gene probes indicate that these environmental isolates not only are nontoxigenic, but also do not possess any of the genetic information encoding cholera toxin, although some isolates from diarrheal stools do.

The cholera vibrios cause many distinctive reactions. They are oxidase positive. The O group 1 cholera vibrios almost always fall into the Heiberg I fermentation pattern that is, they ferment sucrose and mannose but not arabinose, and they produce acid but not gas. Vibrio cholerae also possesses lysine and ornithine decarboxylase, but not arginine dihydrolase. Freshly isolated agar-grown vibrios of the El Tor biotype, in contrast to classical V cholerae, produce a cell-associated mannose-sensitive hemagglutinin active on chicken erythrocytes. This activity is readily detected in a rapid slide test. In addition to hemagglutination, numerous tests have been proposed to differentiate the classical and El Tor biotypes, including production of a hemolysin, sensitivity to selected bacteriophages, sensitivity to polymyxin, and the Voges-Proskauer test for acetoin. El Tor vibrios originally were defined as hemolytic. They differed in this characteristic from classical cholera vibrios however, during the most recent pandemic, most El Tor vibrios (except for the recent isolates from Texas and Louisiana) had lost the capacity to express the hemolysin. Most El Tor vibrios are Voges-Proskauer positive and resistant to polymyxin and to bacteriophage IV, whereas classical vibrios are sensitive to them. As both biotypes cause the same disease, these characteristics have only epidemiologic significance. Strains of the El Tor biotype, however, produce less cholera enterotoxin, but appear to colonize intestinal epithelium better than vibrios of the classical variety. Also, they seem some what more resistant to environmental factors. Thus, El Tor strains have a higher tendency to become endemic and exhibit a higher infection-to-case ratio than the classical biotype.


Recent studies with laboratory animal models and human volunteers have provided a detailed understanding of the pathogenesis of cholera. Initial attempts to infect healthy American volunteers with cholera vibrios revealed that the oral administration of up to 10 11 living cholera vibrios rarely had an effect in fact, the organisms usually could not be recovered from stools of the volunteers. After the administration of bicarbonate to neutralize gastric acidity, however, cholera diarrhea developed in most volunteers given 10 4 cholera vibrios. Therefore, gastric acidity itself is a powerful natural resistance mechanism. It also has been demonstrated that vibrios administered with food are much more likely to cause infection.

Cholera is exclusively a disease of the small bowel. To establish residence and multiply in the human small bowel (normally relatively free of bacteria because of the effective clearance mechanisms of peristalsis and mucus secretion), the cholera vibrios have one or more adherence factors that enable them to adhere to the microvilli (Fig. 24-3). Several hemagglutinins and the toxin-coregulated pili have been suggested to be involved in adherence but the actual mechanism has not been defined. In fact, there may be multiple mechanisms. The motility of the vibrios may affect virulence by enabling them to penetrate the mucus layer. They also produce mucinolytic enzymes, neuraminidase, and proteases. The growing cholera vibrios elaborate the cholera enterotoxin (CT or choleragen), a polymeric protein (Mr 84,000) consisting of two major domains or regions. The A region (Mr 28,000), responsible for biologic activity of the enterotoxin, is linked by noncovalent interactions with the B region (Mr 56,000), which is composed of five identical noncovalently associated peptide chains of Mr 11,500. The B region, also known as choleragenoid, binds the toxin to its receptors on host cell membranes. It is also the immunologically dominant portion of the holotoxin. The structural genes that encode the synthesis of CT reside on a transposon-like element in the V cholerae chromosome, in contrast to those for the heat-labile enterotoxins (LTs) of E coli (Ch. 25), which are encoded by plasmids. The amino acid sequences of these structurally, functionally, and immunologically related enterotoxins are very similar. Their differences account for the differences in physicochemical behavior and the antigenic distinctions that have been noted. There are at least two antigenically related but distinct forms of cholera enterotoxin, called CT-1 and CT-2. Classical O1 V cholerae and the Gulf Coast El Tor strains produce CT-1 whereas most other El Tor strains and O139 produce CT-2. Vibrio cholerae exports its enterotoxin, whereas the E coli LTs occur primarily in the periplasmic space. This may account for the reported differences in severity of the diarrheas caused by these organisms.

Figure 24-3

Vibrio cholerae attachment and colonization in experimental rabbits. The events are assumed to be similar in human cholera. (A) Scanning electron microscopy during early infection. Curved vibrios adhering to epithelial surface. (Approximately × (more. )

Studies in adult American volunteers have shown that 5µ g of CT, administered orally with bicarbonate, causes 1 to 6 L of diarrhea 25µg causes more than 20 L.

Synthesis of CT and other virulence-associated factors such as toxin-coregulated pili are believed to be regulated by a transcriptional activator, Tox R, a transmembrane DNA-binding protein.

The molecular events in these diarrheal diseases involve an interaction between the enterotoxins and intestinal epithelial cell membranes (Fig. 24-4). The toxins bind through region B to a glycolipid, the GM1 ganglioside, which is practically ubiquitous in eukaryotic cell membranes. Following this binding, the A region, or a major portion of it known as the A1 peptide (Mr 21,000), penetrates the host cell and enzymatically transfers ADP-ribose from nicotinamide adenine dinucleotide (NAD) to a target protein, the guanosine 5′-triphosphate (GTP)-binding regulatory protein associated with membrane-bound adenylate cyclase. Thus, CT (and LT) resembles diphtheria toxin in causing transfer of ADP-ribose to a substrate. With diphtheria toxin, however, the substrate is elongation factor 2 and the result is cessation of host cell protein synthesis. With CT, the ADP-ribosylation reaction essentially locks adenylate cyclase in its “on mode” and leads to excessive production of cyclic adenosine 5 1 -monophosphate (cAMP). Pertussis toxin, another ADP-ribosyl transferase, also increases cAMP levels, but by its effect on another G-protein, Gi (Fig. 24-5). The subsequent cAMP-mediated cascade of events has not yet been delineated, but the final effect is hypersecretion of chloride and bicarbonate followed by water, resulting in the characteristic isotonic voluminous cholera stool. In hospitalized patients, this can result in losses of 20 L or more of fluid per day. The stool of an actively purging, severely ill cholera patient can resemble rice water—the supernatant of boiled rice. Because the stool can contain 10 8 viable vibrios per ml, such a patient could shed 2 × 10 12 cholera vibrios per day into the environment. Perhaps by production of CT, the cholera vibrios thus ensure their survival by increasing the likelihood of finding another human host. Recent evidence suggests that prostaglandins may also play a role in the secretory effects of cholera enterotoxin. Recent studies in volunteers using genetically-engineered Tox – strains of V cholerae have revealed that the vibrios have putative mechanisms in addition to CT for causing (milder) diarrheal disease. These include Zot (for Zonula occludens toxin) and Ace (for accessory cholera enterotoxin), and perhaps others, but their role has not been established conclusively. Certainly CT is the major virulence factor and the act of colonization of the small bowel may itself elicit an altered host response (e.g., mild diarrhea), perhaps by a trans-membrane signaling mechanism.

Figure 24-4

Mechanism of action of cholera enterotoxin. Cholera toxin approaches target cell surface. B subunits bind to oligosaccharide of GM1 ganglioside. Conformational alteration of holotoxin occurs, allowing the presentation of the A subunit to cell surface. (more. )

Figure 24-5

Comparison of activities of cholera enterotoxin (CT) with pertussis toxin (PT). The α-subunits of Gs and Gi, with GTP-binding sites, are ADP-ribosylated, respectively, by A1 peptide of CT or by the A subunit of PT, preventing, respectively, the hydrolysis (more. )

Various animal models have been used to investigate pathogenic mechanisms, virulence, and immunity. Ten-day-old suckling rabbits develop a fulminating diarrheal disease after intraintestinal inoculation with virulent V cholerae or CT. Adult rabbits are relatively resistant to colonization by cholera vibrios however, they do respond, with characteristic out pouring of fluid, to the intraluminal inoculation of live vibrios or enterotoxin in surgically isolated ileal loops. Suckling mice are susceptible to intragastric inoculation of vibrios and to orally administered toxin. Adult conventional mice are also susceptible to orally administered toxin, but resist colonization except in isolated intestinal loops. Interestingly, however, germ-free mice can be colonized for months with cholera vibrios. They rarely show adverse effects, although they are susceptible to cholera enterotoxin. Dogs have been used experimentally, although they are relatively refractory and require enormous inocula to elicit choleraic manifestations. Chinchillas also are susceptible to diarrhea following intraintestinal inoculation with moderate numbers of cholera vibrios. Infections initiated by extraintestinal routes of inoculation (e.g., intraperitoneal) largely reflect the toxicity of the lipopolysaccharide endotoxin. The intraperitoneal infection in mice has been used to assay the protective effect of conventional killed vibrio vaccines (no longer widely used).

Various animals, including humans, rabbits, and guinea pigs, also respond to intradermal inoculation of relatively minute amounts of CT with a characteristic delayed (maximum response at 24 hours), sustained (visible up to 1 week or more), erythematous, edematous induration associated with a localized alteration of vascular permeability. In laboratory animals, this response can be measured after injecting a protein-binding dye, such as trypan blue, that extravasates to produce a zone of bluing at the site of intracutaneous inoculation of toxin. This observation has been exploited in the assay of CT and its antibody and in the detection of other enterotoxins.

In addition, because of the broad spectrum of activity of CT on cells and tissues that it never contacts in nature, various in vitro systems can be used to assay the enterotoxin and its antibody. In each, the toxin causes a characteristically delayed, but sustained, activation of adenylate cyclase and increased production of cAMP, and it may cause additional, readily recognizable, morphologic alterations of certain cultured cell lines. The cells most widely used for this purpose are Chinese hamster ovary (CHO) cells, which elongate in response to picogram doses of the toxin, and mouse Y-l adrenal tumor cells, which round up. Cholera toxin has become an extremely valuable experimental probe to identify other cAMP-mediated responses. It also activates adenylate cyclase in pigeon erythrocytes, a procedure that was used by D. Michael Gill to define its mode of action.

These assays and models also have been applied in the study of an expanding number of CT-related and unrelated enterotoxins. These include the LTs of E coli, which are structurally and immunologically similar to it and are effective in any model that is responsive to CT. The family of small molecular weight heat-stable enterotoxins (ST) of E coli, which activate guanylate cyclase, and which are rapidly active in the infant mouse and certain other intestinal models, are clearly unrelated to CT. CT-related enterotoxins have been reported from certain nonagglutinable (non-O group I) Vibrio strains and a Salmonella enterotoxin was shown to be related immunologically to CT. CT-like factors from Shigella and V parahaemolyticus have thus far been demonstrated only in sensitive cell culture systems. Other enterotoxins and enterocytotoxins, which elicit cytotoxic effects on intestinal epithelial cells, also have been described from Escherichia, Klebsiella, Enterobacter, Citrobacter, Aeromonas, Pseudomonas, Shigella, V parahaemolyticus, Campylobacter, Yersinia enterocolitica, Bacillus cereus, Clostridium perfringens, C difficile, and staphylococci. Escherichia coli, some vibrio strains, and some other enteric bacteria produce cytotoxins that, like Shiga toxin of Shigella dysenteriae, act on Vero (African green monkey kidney) cells in vitro. These toxins have been called Shiga-like toxins, Shiga toxin-like toxins, Vero toxins, and Vero cytotoxins. The classic staphylococcal enterotoxins perhaps should more properly be called neurotoxins, as they seem to affect the central nervous system rather than the gut directly to cause fluid secretion or histopathologic effects.

Host Defenses

Infection with cholera vibrios results in a spectrum of responses. These range from no observed manifestations except perhaps a serologic response ( the most common) to acute purging, which must be treated by hospitalization and fluid replacement therapy this is the classic response. The reasons for these differences are not entirely clear, although it is known that individuals differ in gastric acidity and that hypochlorhydric individuals are most prone to cholera. Whether individuals differ in the availability of intestinal receptors for cholera vibrios or for their toxin has not been established. Prior immunologic experience of subjects at risk is certainly a major factor. For example, in heavily endemic regions such as Bangladesh, the attack rate is relatively low among adults in comparison with children. In neoepidemic areas, cholera is more frequent among the working adult population. Resistance is related to the presence of circulating antibody and, perhaps more importantly, local immunoglobulin A (IgA) antibody against the cholera bacteria or the cholera enterotoxin or both. Intestinal IgA antibody can prevent attachment of the vibrios to the mucosal surface and neutralize or prevent binding of the cholera enterotoxin. For reasons that are not clear, individuals of blood group O are slightly more susceptible to cholera. Breastfeeding is highly recommended as a means of increasing immunity of infants to this and other diarrheal disease agents.

Recovery from cholera probably depends on two factors: elimination of the vibrios by antibiotics or the patient's own immune response, and regeneration of the poisoned intestinal epithelial cells. Treatment with a single 200-mg dose of doxycycline has been recommended. As studies in volunteers demonstrated conclusively, the disease is an immunizing process. Patients who have recovered from cholera are solidly immune for at least 3 years.

Cholera vaccines consisting of killed cholera bacteria administered parenterally have been used since the turn of the century. However, recent controlled field studies indicate that little, if any, effective immunity is induced in immunologically virgin populations by such vaccines, although they do stimulate preexisting immunity in the adult population in heavily endemic regions. Controlled studies have likewise shown that a cholera toxoid administered parenterally was ineffective in preventing cholera. Probably the natural disease should be simulated to induce truly effective immunity although a parenterally administered conjugate vaccine consisting of the polysaccharide of the vibrio LPS covalently linked to cholera toxin has given promising results in preliminary studies. Studies in volunteers have shown that orally administered, chemically mutagenized or genetically engineered mutants which do not produce CT or produce only its B subunit protein can induce immunity against subsequent challenge. However, most of these candidate vaccines also produce unacceptable side effects—primarily mild to moderate diarrhea. An exception is strain CVD103-HgR (a mercury resistant A – B + derivative of classical biotype Inaba serotype strain 569B). This strain has minimal reactogenicity but does not colonize well and therefore has to be given in higher doses. Field studies with this strain are in progress. Combined preparations of bacterial somatic antigen and toxin antigen have been reported to act synergistically in stimulating immunity in laboratory animals that is, the combined protective effect is closer to the product than to the sum of the individual protective effects. However, a large field study evaluating such nonviable oral vaccines in Bangladesh revealed that neither the whole-cell bacterin nor the killed vibrios supplemented with the B-subunit protein of the cholera enterotoxin induced sufficient long term protection, especially in children, to justify their recommendation for public health use. No clear-cut advantage of the inclusion of the B-subunit was demonstrated.

In any case, even if these vaccines were effective, the requirement for large and repeated doses would make them too expensive for use in the developing areas that are usually afflicted with epidemic cholera. Moreover, they were clearly less effective in children—the primary target population in heavily endemic areas. Neither the killed whole cell vaccine nor strain CVD103-HgR could be expected to protect against the new O139 serovar.


Humans apparently are the only natural host for the cholera vibrios. Cholera is acquired by the ingestion of water or food contaminated with the feces of an infected individual. Previously, the disease swept the world in six great pandemics and later receded into its ancestral home in the Indo-Pakistani subcontinent. In 1961, the El Tor biotype (a subset distinguished by physiologic characteristics) of V cholerae, not previously implicated in widespread epidemics, emerged from the Celebes (now Sulawesi), causing the seventh great cholera pandemic. In the course of their migration, the El Tor biotype cholera vibrios virtually replaced V cholerae of the classic biotype that formerly was responsible for the annual cholera epidemics in India and East Pakistan (now Bangladesh). The pandemic that began in 1961 is now heavily seeded in Southeast Asia and in Africa. It has also invaded Europe, North America, and Japan, where the outbreaks have been relatively restricted and self-limited because of more highly developed sanitation. Several new cases were reported in Texas in 1981 and sporadic cases have since been reported in Louisiana and other Gulf Coast areas. This now endemic focus appears to be due to a clone which is unique from the pandemic strain. In 1991, the pandemic strain hit Peru with massive force and has since spread through most of the Western Hemisphere, causing more than a million cases. Fortunately, mortality has been less than 1 percent because of the effectiveness of oral rehydration therapy. The vibrios surprised us again, in 1992, with the emergence of O139 in India and Bangladesh. For a while it appeared that O139 would replace O1 (both classical and El Tor) but it has exhibited quiescent periods when O1 reemerges.

Cholera appears to exhibit three major epidemiologic patterns: heavily endemic, neoepidemic (newly invaded, cholera-receptive areas), and, in developed countries with good sanitation, occasional limited outbreaks. These patterns probably depend largely on environmental factors (including sanitary and cultural aspects), the prior immune status or antigenic experience of the population at risk, and the inherent properties of the vibrios themselves, such as their resistance to gastric acidity, ability to colonize, and toxigenicity. In the heavily endemic region of the Indian subcontinent, cholera exhibits some periodicity this may vary from year to year and seasonally, depending partly on the amount of rain and degree of flooding. Because humans are the only reservoirs, survival of the cholera vibrios during interepidemic periods probably depends on a relatively constant availability of low-level undiagnosed cases and transiently infected, asymptomatic individuals. Long-term carriers have been reported but are extremely rare. The classic case occurred in the Philippines, where 𠇌holera Dolores” harbored cholera vibrios in her gallbladder for 12 years after her initial attack in 1962. Her carrier state resolved spontaneously in 1973 no secondary cases had been associated with her well-marked strain. Recent studies, however, have suggested that cholera vibrios can persist for some time in shellfish, algae or plankton in coastal regions of infected areas and it has been claimed that they can exist in 𠇊 viable but nonculturable state.”

During epidemic periods, the incidence of infection in communities with poor sanitation is high enough to frustrate the most vigorous epidemiologic control efforts. Although transmission occurs primarily through water contaminated with human feces, infection also may be spread within households and by contaminated foods. Thus, in heavily endemic regions, adequate supplies of pure water may reduce but not eliminate the threat of cholera.

In neoepidemic cholera-receptive areas, vigorous epidemiologic measures, including rapid identification and treatment of symptomatic cases and asymptomatically infected individuals, education in sanitary practices, and interruption of vehicles of transmission (e.g., by water chlorination), may be most effective in containing the disease. In such situations, spread of cholera usually depends on traffic of infected human beings, although spread between adjacent communities can occur through bodies of water contaminated by human feces. John Snow was credited with stopping an epidemic in London, England, by the simple expedient of removing the handle of the 𠇋road Street pump” (a contaminated water supply) in 1854, before acceptance of the “germ theory” and before the first isolation of the “Kommabacillus” by Robert Koch.

In such developed areas as Japan, Northern Europe, and North America, cholera has been introduced repeatedly in recent years, but has not caused devastating outbreaks however, Japan has reported secondary cases and, in 1978, the United State experienced an outbreak of about 12 cases in Louisiana. In that outbreak, sewage was infected, and infected shellfish apparently were involved. Interestingly, the hemolytic vibrio strain implicated was identical to one that caused an unexplained isolated case in Texas in 1973.


Rapid bacteriologic diagnosis offers relatively little clinical advantage to the patient with secretory diarrhea, because essentially the same treatment (fluid and electrolyte replacement) is employed regardless of etiology. Nevertheless, rapid identification of the agent can profoundly affect the subsequent course of a potential epidemic outbreak. Because of their rapid growth and characteristic colonial morphology, V cholerae can be easily isolated and identified in the bacteriology laboratory, provided, first, that the presence of cholera is suspected and, second, that suitable specific diagnostic antisera are available. The vibrios are completely inhibited or grow somewhat poorly on usual enteric diagnostic media (MacConkey agar or eosin-methylene blue agar). An effective selective medium is thiosulfate-citrate-bile salts-sucrose (TCBS) agar, on which the sucrose-fermenting cholera vibrios produce a distinctive yellow colony. However, the usefulness of this medium is limited because serologic testing of colonies grown on it occasionally proves difficult, and different lots vary in their productivity. This medium is also useful in isolating V parahaemolyticus. They can also be isolated from stool samples or rectal swabs from cholera cases on simple meat extract (nutrient) agar or bile salts agar at slightly alkaline pH values. Following observation of characteristic colonial morphology with a stereoscopic microscope using transmitted oblique illumination, microorganisms can be confirmed as cholera vibrios by a rapid slide agglutination test with specific antiserum. Classic and El Tor biotypes can be differentiated at the same time by performing a direct slide hemagglutination test with chicken erythrocytes: all freshly isolated agar-grown El Tor vibrios exhibit hemagglutination all freshly isolated classic vibrios do not. In practice, this can be accomplished with material from patients as early as 6 hours after streaking the specimen in which the cholera vibrios usually predominate. However, to detect carriers (asymptomatically infected individuals) and to isolate cholera vibrios from food and water, enrichment procedures and selective media are recommended. Enrichment can be accomplished by inoculating alkaline (pH 8.5) peptone broth with the specimen and then streaking for isolation after an approximate 6-hour incubation period this process both enables the rapidly growing vibrios to multiply and suppresses much of the commensal microflora.

The classic case of cholera, which includes profound secretory diarrhea and should evoke clinical suspicion, can be diagnosed within a few minutes in the prepared laboratory by finding rapidly motile bacteria on direct, bright-field, or dark-field microscopic examination of the liquid stool. The technician can then make a second preparation to which a droplet of specific anti-V cholerae O group 1 antiserum is added. This quickly stops vibrio motility. Another rapid technique is the use of fluorescein isothiocyanate-labeled specific antiserum (fluorescent antibody technique) directly on the stool or rectal swab smear or on the culture after enrichment in alkaline peptone broth. For cultural diagnosis, both nonselective and selective (TCBS) media may be used. Although demonstration of typical agglutination essentially confirms the diagnosis, additional conventional tests such as oxidase reaction, indole reaction, sugar fermentation reactions, gelatinase, lysine, arginine, and ornithine decarboxylase reactions may be helpful. Tests for chicken cell hemagglutination, hemolysis, polymyxin sensitivity, and susceptibility to phage IV are useful in differentiating the El Tor biotype from classic V cholerae. Tests for toxigenesis may be indicated.

Diagnosis can be made retrospectively by confirming significant rises in specific serum antibody titers in convalescents. For this purpose, conventional agglutination tests, tests for rises in complement-dependent vibriocidal antibody, or tests for rises in antitoxic antibody can be employed. Convenient microversions of these tests have been developed. Passive hemagglutination tests and enzyme-linked immunosorption assays (ELISAs) have also been proposed.

Cultures that resemble V cholerae but fail to agglutinate in diagnostic antisera (nonagglutinable or non-O group 1 vibrios) present more of a problem and require additional tests such as oxidase, decarboxylases, inhibition by the vibriostatic pteridine compound 0/129, and the “string test.” The string test demonstrates the property, shared by most vibrios and relatively few other genera, of forming a mucus-like string when colony material is emulsified in 0.5 percent aqueous sodium deoxycholate solution. Additional tests for enteropathogenicity and toxigenesis may be useful. Genetically based tests such as PCR are increasingly being used in specialized laboratories.


Treatment of cholera consists essentially of replacing fluid and electrolytes. Formerly, this was accomplished intravenously, using costly sterile pyrogen-free intravenous solutions. The patient's fluid losses were conveniently measured by the use of buckets, graduated in half-liter volumes, kept underneath an appropriate hole in an army-type cot on which the patient was resting. Antibiotics such as tetracycline, to which the vibrios are generally sensitive, are useful adjuncts in treatment. They shorten the period of infection with the cholera vibrios, thus reducing the continuous source of cholera enterotoxin this results in a substantial saving of replacement fluids and a markedly briefer hospitalization. Note, however, that fluid and electrolyte replacement is all-important patients who are adequately rehydrated and maintained will virtually always survive, and antibiotic treatment alone is not sufficient.

Recently it has been recognized that almost all cholera patients and others with similar severe secretory diarrheal disease can be maintained by fluids given orally if the solutions contain a usable energy source such as glucose. Because of this discovery, packets containing appropriate salts are distributed by such organizations as WHO and UNICEF to cholera-afflicted areas, where they are dissolved in water as needed. One such formulation, called ORS for oral rehydration salts, contains NaCl, 3.5 g KCl,1.5 g NaHCO3, 2.5 g (or trisodium citrate, 2.9 g) and glucose, 20.0 g. This mixture is dissolved in 1 L of water and taken orally in increments. Flavoring may be added. Improved versions of ORS, including rice-based formulations that reduce stool output and can be made at home, have been recommended. Unfortunately, this technique, which will save countless millions of lives in developing countries, has not yet been widely accepted by practicing physicians in developed countries.

The possibility of pharmacologic intervention (e.g., a pill that will stop choleraic diarrhea after it has started), has been considered. Two drugs, chlorpromazine and nicotinic acid, have been effective in experimental animals, although the precise mechanism of action has yet to be defined.

Like smallpox and typhoid, cholera—under natural circumstances𠅊ppears to affect only humans therefore, V cholerae as an etiologic entity could conceivably disappear with the last human infection. Nevertheless, the spectrum of cholera-like diarrheal diseases probably will persist for some time.

Cholera is essentially a disease associated with poor sanitation. The simple application of sanitary principles—protecting drinking water and food from contamination with human feces—would go a long way toward controlling the disease. However, at present, this is not feasible in the underdeveloped areas that are afflicted with epidemic cholera or are considered to be cholera receptive. Meanwhile, development of a vaccine that would effectively prevent colonization and manifestations of cholera would be extremely helpful. As indicated above, such vaccines are presently being tested. Antibiotic or chemotherapeutic prophylaxis is feasible and may be indicated under certain circumstances. It also should be mentioned that the incidence of cholera is significantly higher in formula-fed than in breast-fed babies.

Present information indicates that V parahaemolyticus enteritis could be almost completely prevented by applying appropriate procedures to prevent multiplication of the organisms in contaminated seafood, such as keeping it refrigerated continually.

At the Institut Pasteur

The Vibrios and Cholera National Reference Center (CNR), hosted in the Institut Pasteur's Enteric Bacterial Pathogens Unit, has been tasked by the General Directorate of Health in the French Health Ministry with monitoring, confirming and reporting cases of cholera imported into France (there are around 4 or 5 each year). As in many countries, cholera is a notifiable disease in France. The CNR works with microbiologists in countries affected by cholera outbreaks and with non-governmental humanitarian organizations, and is therefore involved in monitoring the strains of V. cholerae in circulation worldwide and in reporting the emergence of new variants or multiple-antibiotic-resistant strains. A genome database recently developed by the unit that traces the history of the seventh cholera pandemic in Africa and Latin America is a valuable tool for improving our understanding of cholera epidemiology.

The Institut Pasteur is a member of WHO's Global Task Force on Cholera Control (GTFCC), a network of over 50 organizations that has adopted a comprehensive multi-sector approach, bringing together multiple partners working to tackle cholera. The Institut Pasteur also leads a Surveillance Working Group, which has published various technical notes. In October 2017, 35 GTFCC partners, including the Institut Pasteur, made an unprecedented commitment to fight cholera by implementing a Global Roadmap (Declaration on Ending Cholera) designed to reduce cholera deaths by 90% by 2030.

Watch the video: Vibrio cholerae Laboratory diagnosis of vibrio cholerae (January 2023).