CHOLERA

INTRODUCTION:

Cholera is an acute, diarrheal illness caused by infection of the intestine with the bacterium Vibrio cholerae. The infection is often mild or without symptoms, but sometimes it can be severe. Approximately one in 20 infected persons has severe disease characterized by profuse watery diarrhea, vomiting, and leg cramps. In these persons, rapid loss of body fluids leads to dehydration and shock. Without treatment, death can occur within hours.

 A person may get cholera by drinking water or eating food contaminated with the cholera bacterium. In an epidemic, the source of the contamination is usually the feces of an infected person. The disease can spread rapidly in areas with inadequate treatment of sewage and drinking water.

The cholera bacterium may also live in the environment in brackish rivers and coastal waters. The disease is not likely to spread directly from one person to another; therefore, casual contact with an infected person is not a risk for becoming ill.

Cholera has been very rare in industrialized nations for the last 100 years; however, the disease is still common today in other parts of the world, including the Indian subcontinent and sub-Saharan Africa.

Cholera can be simply and successfully treated by immediate replacement of the fluid and salts lost through diarrhea. Patients can be treated with oral rehydration solution, a prepackaged mixture of sugar and salts to be mixed with water and drunk in large amounts. This solution is used throughout the world to treat diarrhea. Severe cases also require intravenous fluid replacement. With prompt rehydration, less than 1% of cholera patients die.

Antibiotics shorten the course and diminish the severity of the illness, but they are not as important as rehydration. Persons who develop severe diarrhea and vomiting in countries where cholera occurs should seek medical attention promptly

MICROBIOLOGICAL CHARACTERS

Vibrio cholerae and Asiatic Cholera

The genus Vibrio consists of Gram-negative straight or curved rods, motile by means of a single polar flagellum. Vibrios are capable of both respiratory and fermentative metabolism. O₂ is a universal electron acceptor; they do not denitrify. Most species are oxidase-positive. In most ways vibrios are related to enteric bacteria, but they share some properties with pseudomonads a well. The Family Vibrionaceae is found in the "Facultatively Anaerobic Gram-negative Rods" in Bergey's Manual, on the level with the Family Enterobacteriaceae. Vibrios are distinguished from enterics by being oxidase-positive and motile by means of polar flagella. Of the vibrios that are clinically significant to humans, Vibrio cholerae, the agent of cholera, is the most important.

Most vibrios have relatively simple growth factor requirements and will grow in synthetic media with glucose as a sole source of carbon and energy. However, since vibrios are typically marine organisms, most species require 2-3% NaCl or a sea water base for optimal growth. Vibrios vary in their nutritional versatility, but some species will grow on more than 150 different organic compounds as carbon and energy sources, occupying the same level of metabolic versatility as Pseudomonas. In liquid media vibrios are motile by polar flagella that are enclosed in a sheath continuous with the outer membrane of the cell wall. On solid media they may synthesize numerous lateral flagella which are not sheathed.

Vibrios are one of the most common organisms in surface waters of the world. They occur in both marine and freshwater habitats and in associations with aquatic animals. Some species are bioluminescent and live in mutualistic associations with fish and other marine life. Other species are pathogenic for fish, eels, and frogs, as well as other vertebrates and invertebrates.

Campylobacter jejuni (formerly Vibrio fetus), is now considered to belong in the family Spirillaceae rather than in the family Vibrionaceae. Campylobacter jejuni has been associated with dysentery-like gastroenteritis, as well as with other types of infection, including bacteremic and central nervous system infections in humans. Another vibrio-like organism, Helicobacter pylori causes duodenal and gastric ulcers and gastric cancer.

V. cholerae and V. parahaemolyticus are pathogens of humans. Both produce diarrhea, but in ways that are entirely different. V. parahaemolyticus is an invasive organism affecting primarily the colon; V. cholerae is noninvasive, affecting the small intestine through secretion of an enterotoxin.
 

Vibrio cholerae

Cholera (frequently called Asiatic cholera or epidemic cholera) is a severe diarrheal disease caused by the bacterium Vibrio cholerae. Transmission to humans is by water or food. The natural reservoir of the organism is not known. It was long assumed to be humans, but some evidence suggests that it is the aquatic environment.

V. cholerae produces cholera toxin, the model for enterotoxins, whose action on the mucosal epithelium is responsible for the characteristic diarrhea of the disease cholera. In its extreme manifestation, cholera is one of the most rapidly fatal illnesses known. A healthy person may become hypotensive within an hour of the onset of symptoms and may die within 2-3 hours if no treatment is provided. More commonly, the disease progresses from the first liquid stool to shock in 4-12 hours, with death following in 18 hours to several days.

The clinical description of cholera begins with sudden onset of massive diarrhea. The patient may lose gallons of protein-free fluid and associated electrolytes, bicarbonates and ions within a day or two. This results from the activity of the cholera enterotoxin which activates the adenylate cyclase enzyme in the intestinal cells, converting them into pumps which extract water and electrolytes from blood and tissues and pump it into the lumen of the intestine. This loss of fluid leads to dehydration, anuria, acidosis and shock. The watery diarrhea is speckled with flakes of mucus and epithelial cells ("rice-water stool") and contains enormous numbers of vibrios. The loss of potassium ions may result in cardiac complications and circulatory failure. Untreated cholera frequently results in high (50-60%) mortality rates.

Treatment of cholera involves the rapid intravenous replacement of the lost fluid and ions. Following this replacement, administration of isotonic maintenance solution should continue until the diarrhea ceases. If glucose is added to the maintenance solution it may be administered orally, thereby eliminating the need for sterility and IV. Administration. By this simple treatment regimen, patients on the brink of death seem to be miraculously cured and the mortality rate of cholera can be reduced more than ten-fold. Most antibiotics and chemotherapeutic agents have no value in cholera therapy, although a few (e.g. tetracyclines) may shorten the duration of diarrhea and reduce fluid loss.

History and spread of epidemic cholera:

Cholera has smoldered in an endemic fashion on the Indian subcontinent for centuries. There are references to deaths due to dehydrating diarrhea dating back to Hippocrates and Sanskrit writings. Epidemic cholera was described

in 1563 by Garcia del Huerto, a Portuguese physician at Goa, India. The mode of transmission of cholera by water was proven in 1849 by John Snow, a London physician. In 1883, Robert Koch successfully isolated the cholera vibrio from the intestinal discharges of cholera patients and proved conclusively that it was the agent of the disease.

The first long-distance spread of cholera to Europe and the Americas began in 1817 and by the early 20th century, six waves of cholera had spread across the world in devastating epidemic fashion. Since then, until the 1960s, the disease contracted, remaining present only in southern Asia. In 1961, the "El Tor" biotype (distinguished from classic biotypes by the production of hemolysins) reemerged to produce a major epidemic in the Philippines and to initiate a seventh global pandemic (See map below). Since then this biotype has spread across Asia, the Middle East, Africa, and more recently, parts of Europe.

There are several characteristics of the El Tor strain that confer upon it a high degree of "epidemic virulence" allowing it to spread across the world as previous strains have done. First, the ratio of cases to carriers is much less than in cholera due to classic biotypes (1: 30-100 for El Tor vs. 1: 2 - 4 for "classic" biotypes). Second, the duration of carriage after infection is longer for the El Tor strain than the classic strains. Third, the El Tor strain survives for longer periods in the extraintestinal environment. Between 1969 and 1974, El Tor replaced the classic strains in the heartland of endemic cholera, the Ganges River Delta of India.
 
 

The global spread of cholera during the seventh pandemic, 1961-1971

El Tor broke out explosively in Peru in 1991 (after an absence of cholera there for 100 years), and spread rapidly in Central and South America, with recurrent epidemics in 1992 and 1993. From the onset of the epidemic in January 1991 through September 1, 1994, a total of 1,041,422 cases and 9,642 deaths (overall case-fatality rate: 0.9%) were reported from countries in the Western Hemisphere to the Pan American Health Organization. In 1993, the numbers of reported cases and deaths were 204,543 and 2362, respectively.

So far, the United States has been spared except for imported cases, or clusters of infections from imported food. In the United States during 1993 and 1994, 22 and 47 cholera cases were reported to CDC, respectively. Of these, 65 (94%) were associated with foreign travel.

In 1982, in Bangladesh, a classic biotype resurfaced with a new capacity to produce more severe illness, and it rapidly replaced the El Tor strain which was thought to be well-entrenched. This classic strain has not yet produced a major outbreak in any other country.

In December, 1992, a large epidemic of cholera began in Bangladesh, and large numbers of people have been involved. The organism has been characterized as V. cholerae O139 "Bengal". It is derived genetically from the El Tor pandemic strain but it has changed its antigenic structure such that there is no existing immunity and all ages, even in endemic areas, are susceptible. The epidemic has continued to spread. and V. choleraeO139 has affected at least 11 countries in southern Asia. Specific totals for numbers of V. cholerae O139 cases are unknown because affected countries do not report infections caused by O1 and O139 separately.

Antigenic Variation and LPS Structure in Vibrio cholerae:

Antigenic variation plays an important role in the epidemiology and virulence of cholera. The emergence of the Bengal strain, mentioned above, is an example. The flagellar antigens of V. cholerae are shared with many water vibrios and therefore are of no use in distinguishing strains causing epidemic cholera. O antigens, however, do distinguish strains of V. cholerae

into 139 known serotypes. Almost all of these strains of V. cholerae are nonvirulent. Until the emergence of the Bengal strain (which is "non-O1") a single serotype, designated O1, has been responsible for epidemic cholera. However, there are three distinct O1 biotypes, named Ogawa, Inaba and Hikojima, and each biotype may display the "classical" or El Tor phenotype. The Bengal strain is a new serological strain with a unique O-antigen which partly explains the lack of residual immunity.

Antigenic Determinants of Vibrio cholerae

Endotoxin is present in Vibrio cholerae as in other Gram-negative bacteria. Fewer details of the chemical structure of Vibrio cholerae LPS are known than in the case of E. coli and Salmonella typhimurium, but some unique properties have been described. Most importantly, variations in LPS occur in vivo and in vitro, which may be correlated with reversion in nature of nonepidemic strains to classic epidemic strains and vice versa.

Cholera Toxin:

Cholera toxin activates the adenylate cyclase enzyme in cells of the intestinal mucosa leading to increased levels of intracellular cAMP, and the secretion of H₂0, Na⁺, K⁺, Cl^-, and HCO₃^- into the lumen of the small intestine. The effect is dependent on a specific receptor, monosialosyl ganglioside (GM1 ganglioside) present on the surface of intestinal mucosal cells. The bacterium produces an invasin, neuraminidase, during the colonization stage which has the interesting property of degrading gangliosides to the monosialosyl form, which is the specific receptor for the toxin.

The toxin has been characterized and contains 5 binding (B) subunits of 11,500 daltons, an active (A1) subunit of 23,500 daltons, and a bridging piece (A2) of 5,500 daltons that links A1 to the 5B subunits. Once it has entered the cell, the A1 subunit enzymatically transfers ADP ribose from

NAD to a protein (called Gs or Ns), that regulates the adenylate cyclase system which is located on the inside of the plasma membrane of mammalian cells.

Enzymatically, fragment A1 catalyzes the transfer of the ADP-ribosyl moiety of NAD to a component of the adenylate cyclase system. The process is complex. Adenylate cyclase (AC) is activated normally by a regulatory protein (GS) and GTP; however activation is normally brief because another regulatory protein (Gi), hydrolyzes GTP. The normal situation is described as follows.
 
 

The A1 fragment catalyzes the attachment of ADP-Ribose (ADPR) to the regulatory protein forming Gs-ADPR from which GTP cannot be hydrolyzed. Since GTP hydrolysis is the event that inactivates the adenylate cyclase, the enzyme remains continually activated. This situation can be illustrated as follows.
 
 

  Thus, the net effect of the toxin is to cause cAMP to be produced at an abnormally high rate which stimulates mucosal cells to pump large amounts of Cl^- into the intestinal contents. H₂O, Na⁺ and other electrolytes follow due to the osmotic and electrical gradients caused by the loss of Cl^-. The lost H₂O and electrolytes in mucosal cells are replaced from the blood. Thus, the toxin-damaged cells become pumps for water and electrolytes causing the diarrhea, loss of electrolytes, and dehydration that are characteristic of cholera. 

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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. The A subunit enters the cell. The disulfide bond of the A subunit is reduced by intracellular glutathione, freeing A1 and A2. NAD is hydrolyzed by A1, yielding ADP-ribose and nicotinamide. One of the G proteins of adenylate cyclase is ADP-ribosylated, inhibiting the action of GTPase and locking adenylate cyclase in the "on" mode.

Colonization of the Small Intestine:

There are several characteristics of pathogenic V. cholerae that are important determinants of the colonization process. These include adhesins, neuraminidase, motility, chemotaxis and toxin production. If the bacteria are able to survive the gastric secretions and low pH of the stomach, they are well adapted to survival in the small intestine. V. cholerae is resistant to bile salts and can penetrate the mucus layer of the small intestine, possibly aided by secretion of neuraminidase and proteases. They withstand propulsive gut motility by their own swimming ability and chemotaxis directed against the gut mucosa.

Specific adherence of V. cholerae to the intestinal mucosa is probably mediated by long filamentous fimbriae that form bundles at the poles of the cells. These fimbriae have been termed Tcp pili (for toxin coregulated pili), because expression of these pili genes is coregulated with expression of the cholera toxin genes. Not much is known about the interaction of Tcp pili with host cells, and the host cell receptor for these fimbriae has not been identified. Tcp pili share amino acid sequence similarity with N-methylphenylalanine pili of Pseudomonas and Neisseria.

Two other possible adhesins in V. cholerae are a surface protein that agglutinates red blood cells (hemagglutinin) and a group of outer membrane proteins which are products of the acf (accessory colonization factor) genes. acf mutants have been shown to have reduced ability to colonize the intestinal tract. It has been suggested that V. cholerae might use these nonfimbrial adhesins to mediate a tighter binding to host cells than is attainable with fimbriae alone.

V. cholerae produces a protease originally called mucinase that degrades different types of protein including fibronectin, lactoferrin and cholera toxin itself. Its role in virulence is not known but it probably is not involved in colonization since mutations in the mucinase gene (designated hap for hemagglutinin protease) do not exhibit reduced virulence. It has been suggested that the mucinase might contribute to detachment rather than attachment. Possibly the vibrios would need to detach from cells that are being sloughed off of the mucosa in order to reattach to newly formed mucosal cells.

Regulation of Virulence Factors in Vibrio cholerae:

In Vibrio cholerae, the production of virulence factors is regulated at several levels. Regulation of genes at the transcriptional level, especially the genes for toxin production and fimbrial synthesis, has been studied in the greatest detail.

V. cholerae enterotoxin is a product of ctx genes. ctxA encodes the A subunit of the toxin, and ctxB encodes the B subunit. The genes are part of the same operon. The transcript (mRNA) of the ctx operon has two ribosome binding sites (rbs), one upstream of the A coding region and another upstream of the B coding region. The rbs upstream of the B coding region is at least seven-times stronger than the rbs of the A coding region. In this way the organism is able to translate more B proteins than A proteins, which is required to assemble the toxin in the appropriate 1A: 5B proportion. The components are assembled in the periplasm after translation. Any extra B subunits can be excreted by the cell, but A must be attached to 5B in order to exit the cell. Intact A subunit is not enzymatically active, but must be nicked to produce fragments A1 and A2 which are linked by a disulfide bond. Once the cholera toxin has bound to the GM1 receptor on host cells, the A1 subunit is released from the toxin, presumably by reduction of the disulfide bond that links it to A2, and enters the cell by an unknown translocation mechanism. One hypothesis is that the 5 B subunits form a pore in the host cell membrane through which the A1 unit passes.

Transcription of the ctxAB operon is regulated by a number of environmental signals, including temperature, pH, osmolarity, and certain amino acids. Several other V. cholerae genes are coregulated in the same

manner including the tcp operon which is concerned with fimbrial synthesis and assembly. Thus the ctx operon and the tcp operon are part of a regulon, the expression of which is controlled by the same environmental signals.

The proteins involved in control of this regulon expression have been identified as ToxR, ToxS and ToxT. ToxR is a transmembranous protein with about two-thirds of its amino terminal part exposed to the cytoplasm. ToxR dimers, but not ToxR monomers, will bind to the operator region of ctxAB operon and activate its transcription. ToxS is a periplasmic protein. It is thought that ToxS can respond to environmental signals, change conformation, and somehow influence dimerization of ToxR which activities transcription of the operon. Possibly ToxR and ToxS form a standard two-component regulatory system with ToxS functioning as a sensor protein that phosphorylates and thus converts ToxR to its active DNA binding form. However, no phosphorylation of ToxR has been detected. ToxT is thought to be a cytoplasmic protein that is a transcriptional activator of the tcp operon. Expression of ToxT is activated by ToxR, while ToxT, in turn, activates transcription of tcp genes for synthesis of tcp pili.

Thus, the ToxR protein is a regulatory protein which functions as an inducer (not a repressor) in a system of positive control. Possibly through an interaction with ToxS, it can sense some change in the environment (outside the cell) and transmit a molecular signal to the chromosome which induces the transcription of genes for attachment (pili formation) and toxin production. It is reasonable to expect that the environmental conditions that exist in the GI tract (i.e., 37^o temperature, low pH, high osmolarity, etc.), as opposed to conditions in the extraintestinal (aquatic) environment of the vibrios, are those that are necessary to induce formation of the virulence factors necessary to infect. However, a curious observation of the vibrios made in vitro, that toxin production is turned on at 30 degrees and turned off at 37 degrees, leads to speculation of the ecological function of the toxin during human infection.

Immunity to Cholera:

Infection with V. cholerae results in a spectrum of responses ranging from life-threatening secretory diarrhea to mild or unapparent infections of no manifestation except a serologic response. The reasons for these differences are not known. One idea is that individuals differ in the availability of intestinal receptors for cholera vibrios or for their toxin, but this has not been proven. Prior immunologic experience 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.

After natural infection by V. cholerae, circulating antibodies can be detected against several cholera antigens including the toxin, somatic (O) antigens, and flagellar (H) antigens. All these antibodies can also be raised by parenteral injection of antigens as vaccine components. Antibodies directed against Vibrio O antigens are considered "vibriocidal" antibodies because they will lyse V. cholerae cells in the presence of complement and serum components. Vibriocidal antibodies reach a peak 8-10 days after the onset of clinical illness, and then decrease, returning to the baseline 2 - 7 months later. Their presence correlates with resistance to infection, but they may not be the mediators of this protection and the role of circulating antibodies in natural infection is unclear.

After natural infection, patients also develop toxin-neutralizing antibodies. In a natural setting for cholera, there is no correlation between antitoxic antibody levels and the incidence of disease.

Since cholera is essentially a topical disease of the small intestine, it would seem that topical defense might be a main determinant of protection against infection by V. cholerae. Recurrent infections of cholera are in fact, rare, and this is probably due to local immune defense mediated by antibodies secreted onto the surfaces of the intestinal mucosa.

Secretory IgA, as well as IgG and IgM in serum exudate, can be detected in the intestinal mucosa of immune individuals. Although these antibodies presumably have to function in the absence of complement they still bring about protective immunity. Motility is important in pathogenesis, and antibodies against flagella could immobilize the vibrios. Antibodies against flagella or somatic O antigens could cause clumping and arrested motion of cells. Antitoxic antibodies could react with toxin at the epithelial cell surface and block binding of the toxin. The process to which the vibrios attach to the intestinal epithelium is highly specific and antibodies against Vibrio fimbriae or other surface components (LPS?) could block attachment.

The observation that natural infection confers effective and long-lasting immunity against cholera has led to efforts to develop a vaccine which will elicit protective immunity. The first attempts at a vaccine in 1960s were directed at whole cell preparations injected parenterally. At best, 90% protection was achieved and this immunity waned rapidly to the baseline within one year. Purified LPS fractions from different biotypes have also been given as vaccines with variable success. The cholera toxin can be converted to toxoid in the presence of formalin and glutaraldehyde. The toxoid is a poor antigen, however, and it elicits a very low level of protection. At the present, a good vaccine for cholera does not exist.

Attempts are underway to develop an oral vaccine from a live attenuated strain of V. cholerae. The ideal properties for such a strain would be to have all the pathogenicity factors required for colonization of the small intestine (motility, fimbriae, neuraminidase, etc.) but not to produce a complete toxin molecule. Ideally it should produce only the B subunit of the toxin which would stimulate formation of antibodies that could neutralize the binding of the native toxin molecule to epithelial cells.

A new vaccine has been developed to combat the Vibrio cholerae Bengal strain that has started spreading in epidemic fashion in the Indian subcontinent and Southeast Asia. The Bengal strain differs from previously isolated epidemic strains in that it is serogroup 0139 rather than 01, and it expresses a distinct polysaccharide capsule. Since previous exposure to 01 Vibrio cholerae does not provide protective immunity against 0139, there is no residual immunity in the indigenous population to the Bengal form of cholera.

The noncellular vaccine is relatively nontoxic and contains little or no LPS and other impurities. The vaccine will be used for active immunization against Vibrio cholerae Bengal and other bacterial species expressing similar surface polysaccharides. In addition, human or other antibodies induced by this vaccine could be used to identify Vibrio cholerae Bengal for the diagnosis of the infection and for environmental monitoring of the bacterium.

Tailpiece:

E. coli produces a toxin, heat labile toxin (LT), that is very similar to the cholera toxin in structure and mode of action. The DNA that encodes the LT toxin is on a plasmid that can be transferred to other E. coli strains and probably to other enteric bacteria, as well. Close relationships between the genetic code for LT toxin and the cholera toxin undoubtedly exist but have not been documented as yet. The genetic information for the toxin in V. cholerae is located on the bacterial chromosome. Other bacterial enterotoxins related to cholera toxin have been reported in non-group O

Vibrio strains and a strain of Salmonella .

Clinical Presentation of Cholera:

Most cholera infections are asymptomatic or mild, and indistinguishable from other mild diarrhoea. In its severe form the following signs and symptoms characterise cholera:

• Onset is typically sudden,
• Diarrhoea is profuse, painless and watery, with flecks of mucus in the stool ("rice water" stools). The presence of blood in stools is not a characteristic of cholera,
• Vomiting may occur, usually early in the illness,
• Majority of patients are afebrile, children are more often febrile than adults,
• Dehydration occurs rapidly (up to 1000ml/hour of diarrhoea may be produced),
• All complications result from effects of loss of fluids and electrolytes in stool; and

• Vomitus; muscle cramps, acidosis, peripheral vasoconstriction, and ultimately renal and circulatory failure, arrhythmias and death may occur if treatment is not given timeously.

Reservoir:

For practical purposes, cholera is restricted to humans. Faecally contaminated water is the most important reservoir of infection and vehicle of transmission, either directly or indirectly through contaminated food.

Mode of Transmission:

Vibrio cholerae is spread mainly via the faecal-oral route. Some of the best-known sources of infection are as follows:

• Drinking water that has been contaminated at its source, during storage or usage,
• Contaminated foods, vegetables that have been fertilised with human excreta (nightsoil) or "freshened" with contaminated water,
• Soiled hands can also contaminate clean drinking water and food, and
• Fish, particularly shellfish taken from contaminated water and eaten raw or insufficiently cooked.

Incubation Period:

The incubation period ranges form a few hours to 5 days, (usually 2 - 3 days).

Period of Communicability:

Cholera is communicable in the duration of stool-positive stage. Asymptomatic carrier status may persist for several months.

Population at Risk:

The people most at risk of contracting cholera are those who do not have access to piped safe water and adequate and proper sanitation.

Clinical Features:

The severity of cholera is dependent on the rapidity and duration of fluid loss.  However, a typical case of cholera shows three stages.

Stages of Cholera:

Ø      Stage of Evacuation

Ø      Stage of Collapse

Ø      Stage of Recovery

            Stage of Evacuation:

            The onset is abrupt with profuse, painless, watery diarrhoea followed     by vomiting.  The patient may pass as many as 40 stools in a day.  The         stools may have “rice water” appearance.

            Stage of Collapse:

            The patient soon passes into a stage of collapse because of        dehydration.  The classical signs are: sunken eyes, hollow cheeks, scaphoid abdomen, sub normal temperature, washerman hands and       feet, absent pulse, unrecordable blood pressure, loss of skin elasticity,    shallow and quick respirations. The output of urine decreases and may   cease. The patient becomes restless and complains of intense thirst and cramps in legs and abdomen.  Death may occur at this stage, due       to dehydration and acidosis resulting from diarrhea.

        Stage of recovery:

            If death does not occur, the patient begins to show signs of clinical         improvement.

v     The blood pressure begins to rise. 

v     The temperature returns to normal.

v     Urine secretion is reestablished, if anuria persists, the patient may die of renal failure.

The classical form of severe cholera occurs only 5-10% of cases.  In the rest, the disease tends to be mild characterized by diarrhea with or without vomiting are marked dehydration.  As a rule mild cases recover in 1to 3 days.

EPIDEMIC PREPAREDNESS:

A strong programme for the control of diarrhoeal diseases is the best preparation for a cholera epidemic. In the long term, improvements of safe water supply and adequate sanitation are the best means of preventing cholera. In an outbreak, the best control measures are the early detection of cases and treatment of patients; coupled with health education. In order to respond quickly to the cholera epidemic and to prevent deaths, health facilities must have access to adequate quantities of essential supplies, particularly oral rehydration solution and intravenous fluids.

During the outbreak of cholera, these supplies are needed in greater quantities than normal. To prepare for an outbreak, it is essential to maintain additional stocks at appropriate points in the drug delivery system. Small 'buffer stocks" should be placed at local health facilities, larger buffer stocks at district or provincial levels, and an adequate emergency stock at a central distribution point.

Medical and paramedical personnel involved in the treatment of cholera should receive intensive and continuing training to ensure that they are familiar with the most effective techniques for the management of patients with cholera.

PREVENTION AND CONTROL:

• In approximately 90% of cholera cases, the disease is mild; and it is difficult to differentiate it from other diarrhoeal diseases;
• Oral rehydration therapy is important in case management and can reduce the case fatality;
• Vaccination and other chemoprophylaxis are ineffective in preventing and controlling cholera; and personal hygiene on drinking and eating habits, safe disposal of human waste have proven to be effective in controlling the disease.

Cholera epidemics are public health problems and could claim up to 50% of its victims. It is therefore important for all the stakeholders in cholera prevention and control to use correct intervention strategies useful in curbing the epidemic.

All travelers to areas where cholera has occured should observe the following recomendations:

• Drink only water that you have boiled or treated with chlorine or iodine. Other safe beverages include tea and coffee made with boiled
  water and carbonated, bottled beverages with no ice.
• Eat only foods that have been thoroughly cooked and are still hot, or fruit that you have peeled yourself.
• Make sure all vegetables are cookedavoid salads.
• Avoid foods and beverages from street vendors.

A simple rule of thumb is "Boil it, cook it, peel it, or forget it."

Preventive Measures:

The community should be informed about sources of contamination and ways to avoid infection. Attention to sanitation can markedly reduce the risk of transmission of cholera including other intestinal pathogens. This is especially true where lack of good sanitation may lead to contamination of water sources. High priority should be given to observing the basic principles of sanitary human waste disposal and particularly the protection of water sources from faecal contamination.

The development of sanitary systems appropriate to local conditions should be facilitated and their siting in relation to water sources emphasised. Basic hygiene involving thorough hand washing following contact with excreta should be encouraged for adults, infants and children.

Where water supplies are at risk of contamination, households should be taught about the necessity and the techniques of sanitising water in the home. The simplest and most cost effective method is chlorination of water in the storage container using household bleach. Boiling is also effective. Filtration may be necessary in addition to boiling if the only water available contains much particulate matter. Chlorination alone is not sufficient in such circumstances. Even when drinking water is rendered safe, infection may still be transmitted by contaminated surface water used for bathing and for washing clothing, food or cooking utensils. In an outbreak situation all water

sources with potential for contamination must be tested, rendered safe if contaminated or otherwise closed to usage and alternative sources provided.

Since food is an important vehicle for the transmission of enteric pathogens, attention to food safety is an essential preventive measure, which should be intensified when there is a threat of cholera. Street vendors and communal food sources will require particular attention, since they pose a special risk. Flies play a relatively small role in spreading cholera but their presence in large numbers indicates poor sanitary conditions, which favour transmission of the disease.

Public Awareness:

In case of an outbreak, communities at risk should be sensitised through intensive health education; and encouraged to participate in the following activities:

• Water purification or ensuring a safe water supply by boiling or chlorination of domestic water using household bleach: Add 1 teaspoonful (5ml, or one capful if bottle has a screw cap) of household bleach to 20-25 liters of water. Thoroughly mix solution with the water and allow to stand for at least two hours (preferably overnight) before use.
• Sanitary disposal of human waste without contaminating water sources and control of flies.
• Food hygiene - avoid any potentially contaminated food especially raw or partially cooked fish and shellfish. Food of vegetable origin should be peeled or shelled. Boil or pasteurise of all milk.

Actively inform and educate health care workers and the community about the extent and severity of the outbreak and the effectiveness and simplicity of current treatment methods, and benefits of reporting cholera cases promptly. The free flow of information would prevent panic spreading through the community. Communities should also be involved in educating themselves through the use of various communication strategies. Street food-vendors and restaurants may contribute in the spread of the disease. Therefore, Environmental Health Officers need to be vigilant in inspecting food-handling practices, and should be authorised to stop street sales or close restaurants if insanitary practices are revealed.

Health education activities for food handlers in areas under the threat of cholera should stress the following:

• Exclude infected persons from handling food
• Wash vegetables and fruit in treated water before use
• Prepare and store food under proper hygienic conditions
• Cook food thoroughly in treated water and eat it while still hot, or reheat it thoroughly before eating
• Prevent contamination of food by contact with other contaminated raw food, contaminated surfaces or flies
• Wash hands thoroughly with soap after defaecation and before preparing or eating food
• Encourage individuals to use clean cutlery when eating
• Discourage the habit of several people eating simultaneously from a communal food container
• Left over food should be reheated before eating
• Encourage breast-feeding of infants.

It is very important to liaise with local media such as press, radio and television to ensure that correct health education messages are passed on to the general public.

Control of Patients, Contacts and Environment:

An organised programme for the control of diarrhoeal diseases is the best preparation for a cholera outbreak. The best control measures are the early detection and effective treatment of infected persons allied to health education. The mortality is likely to be high among severe cases (up to 50%) in an unprepared community.

The basic requirements for preparedness include the establishment of a reliable surveillance and reporting system, ensuring the availability of essential supplies and the training of workers in the clinical management of acute diarrhoea.

Laboratory diagnosis:

Laboratory methods of diagnosis are required to confirm the diagnosis. 

·        Collection of stools: Collection of stools by following ways, rubber catheter, rectal swab.

·        Collection of vomitus is never used

·        Water samples containing 1-3 liters of suspect water should be collected in sterile bottles and despatched to lab by the quick method of transport.

·        Food samples suspected to be contaminated with vibrio cholerae amounting to 1-3 grams and collected in a transport media and sent to lab.

·        Transportation: The stool should be transported by Venkatraman-Ramakrishnan medium or alkaline peptone water or Cary – Blair medium if it is collected by the rectal swab.

·        Direct examination by microscopy with dark field illumination.

·        Culture methods: peptone water tellurite medium for enrichment, sub cultured on bile salt agar medium.

·        Characterization: In BSA medium, V.cholerae appears as translucent, moist, raised, smooth and easily emulsifiable colonies.  The colonies are picked up and tested by Gramstain and Motility, serological test.

·        Biochemical test: production of acid in sucrose and mannose, but not arabinose.

·        Further characterization by the direct haemagglutination test, polymyxin B sensitivity test, sensitivity to cholera phage IV, V-P reaction, haemolysis test.

SURVEILLANCE:

Bacterial Surveillance:

INSTRUCTIONS FOR COLLECTION OF STOOL SPECIMENS FOR CHOLERA INVESTIGATIONS

General information:

1. Specimen labels must be properly filled in. Specimens should be collected before antibiotic treatment.
2. Delays between collection of specimens and dispatch to the laboratory should be minimised.
3. Stools may be sent:

a.       In normal specimen containers for isolation of all pathogens including Vibrio cholerae, or

b.      In single strengths alkaline peptone water, specifically for accelerated Vibrio cholerae isolation. Dip a swab into the stool and express fluid against the inside of the bottle; repeat. Discard swab into disinfectant, or

c.       If a delay of more than 24 hours is anticipated, the specimen should be submitted in Cary-Blair transport medium. Swabs should be plunged deeply into the medium, left in position for at least 30 seconds, then twisted gently and removed.

NOTE: This applies to plastic-stemmed swabs, if wooden-stemmed swabs are used, these can be broken off at the lip of the specimen container after plunging into the transport medium.

5. Specimen containers with alkaline peptone water and Cary-Blair transport medium may be ordered from the South African Institute for Medical Research Stores, Johannesburg or from the laboratory serving the respective hospital clinic.

l proven cases must be reported immediately through the line listing form to the local authority who must report to the Provincial Communicable Disease Control Officer and the National Department of Health. An attempt must be made to establish a bacteriological diagnosis from rectal swabs or stool specimens;  in cases of gastro-enteritis suspected of being due to or possibly due to cholera, presenting at hospitals/peripheral clinics or observed by mobile health teams and field workers in cholera designated areas.

Environmental Surveillance:

Environmental surveillance forms one of the most important part in the control and preparedness of the cholera epidemic. The following are to be taken into consideration when conducting an environmental surveillance.

• Identify communities at risk (unsafe water supplies or inadequate sanitation) and ensure that they are informed about sources of contamination and ways to avoid infection
• Investigate all bacteriologically proven cases to identify the sources of infection
• Monitor the spread of cholera in risk areas by periodically sampling strategic sewage effluent (hospitals, prisons, hostels, sewage purification works) as an early warning system (Annexure B).
• Surveillance using Moore pads should only be done in high-risk areas where there is a definite chance of cholera being identified. Selected sentinel sites should be monitored in large cities. Moore pads should be used to monitor the end on an epidemic. Vibrio cholerae isolated from Moore pads should be tested to determine whether they are indeed Vibrio cholerae 01 or not, since Vibrio cholerae 01 result in diarrhoea.

When such changes in the pattern of diarrhoeal illness occur the notification process should be activated immediately. When this information comes from an area where cholera has.not previously been confirmed, bacteriological and epidemiological investigations should be arranged promptly to establish the cause of the outbreak and epidemic control measures instituted, if indicated.

Reporting:

When suspected cases of cholera are detected at a health facility, the nearest referral facility or designated local health officer should be notified immediately. The Provincial Department of Health should then be notified to investigate and confirm the diagnosis. Upon confirmation, the National Department of Health should be notified since cholera is a notifiable disease.

Either the Provincial or National Department of Health should proactively inform the community via the media, of the cholera threat and measures to be taken to prevent the outbreak from spreading.

The National Communicable Disease Officer should then inform the Senior Management of the outbreak of the disease and the steps being taken to contain and control the outbreak. The opportunity should be used to motivate for improved water and sanitation through provision of safe water supplies and the building of toilets or latrines.

Notification According to International Health Regulations:

According to these regulations National Health Authorities should report the first suspected cases of cholera to the World Health Organisation as rapidly as possible. Laboratory confirmation should be obtained at the earliest opportunity and also reported to WHO. Weekly reporting is required where cholera is confirmed.

Reports should include the number of new cases and deaths since the previous report plus the cumulative totals for the current year by province or other applicable geographic division.

Additional demographic information should be provided, if available. Once the presence of cholera in an area has been confirmed it is not a requirement to confirm all subsequent cases.

Neither the treatment of individual cases nor the notification of suspected cases needs laboratory confirmation of the presence of Vibrio cholerae 01. Monitoring of an epidemic should include laboratory confirmation of a small proportion of cases on a continuing basis.

TREATMENT:

Hospitalisation with enteric precautions is desirable for severely ill patients but strict isolation is not necessary. Less severe cases can be managed on an outpatient basis with oral rehydration. Crowded cholera wards can be operated without hazard to staff and visitors when effective hand washing and basic procedures of cleanliness are practiced. The only treatment needed is rehydration as soon as possible. It is essential that all cases presenting clinically as cholera cases, must be treated as such immediately.

Management of Cholera Patients:

Recognition of cholera cases "rice water stools" is very important, and health workers need to start treatment as early as possible to reduce potential contamination of the environment and death. Cholera should be suspected when:

• A patient older than five years develops severe dehydration from acute watery diarrhoea (usually with vomiting.) or
• Any patient above the age of two years has acute watery diarrhoea in an area where there is an outbreak of cholera.

Rehydration:

Dehydration, acidosis, and potassium depletion typical of cholera are due to loss of water and salts through diarrhoea and vomiting. Therefore rehydration, which consists of replacing water and salts, is necessary. Patients should be encouraged to seek medical attention from trained health workers as rapidly as possible to reduce the risk of shock. To follow are steps useful for the management of cholera patients.

Steps in the Management of Suspected Cholera:

Step 1: Assess dehydration

Step 2: Rehydrate the patient, and monitor frequently, and reassess hydration status

Step 3: Maintain hydration: replace continuing fluid losses until diarrhoea stops.

Step 1: Assess the Patients for Dehydration

Use Table 1 to determine whether the patient has severe, some or no signs of dehydration.

·         In adults and children older than 5 years, other *signs* for severe dehydration are *absent radial pulse* and *low blood pressure). The skin pinch may be less useful in patients with marasmus (severe wasting) or kwashiorkor (severe malnutrition with oedema), or obese patients. Tears are a relevant sign only for infants and young children

Step 2: Rehydrate the Patient, and Monitor Frequently, Reassess Hydration Status

1. For Severe Dehydration:

·         Give IV fluid immediately to replace fluid deficit. Use Ringer's lactate solution or, if not available, normal saline.

·         If the patient can drink give ORS by mouth simultaneously while the drip is being set up.

·         For patients aged 1 year and older, give 100 ml/kg IV in 3 hours, as follows:

·         30 ml/kg as rapidly as possible (within 30 minutes); then

·         70 ml/kg in the next 2,5 hours

·         For patients aged less than 1 year, give 100ml/kg IV in 6 hours, as follows:

·         30 ml/kg in the first hour; then

·         70 ml/kg in the next 5 hours

·         Monitor the patient very frequently. After the initial 30 ml/kg have been given, the radial pulse should be strong and blood pressure should be normal: If the pulse is not yet strong, continue to give IV fluid rapidly

·         Give ORS solution (about 5 ml/kg per hour) as soon as the patient can drink, in addition to IV fluid

·         Reassess the patient after 3 hours (infants after 6 hours), using Table 1:

·         If there are still signs of severe dehydration (this is rare), repeat the IV therapy

·         If there are signs of some dehydration, continue as indicated below for some dehydration

·         If there are no signs of dehydration, go on to step 3 to maintain hydration by replacing continuing fluid losses.

2. For Moderate Dehydration:

·         Give ORS solution in the amount recommended in Table 2. If the patient passes watery stools or wants more ORS solution than shown, give more.

·         Monitor the patient frequently to ensure that ORS solution is taken satisfactorily and to detect patients with profuse and continuing diarrhoea who will require closer monitoring.

·         Reassess the patient after 4 hours, using Table 1:

·         If signs of severe dehydration have appeared (this is rare), treat as in step 1, above.

·         If there is still Moderate dehydration, repeat the procedures for some dehydration, and start to offer food and other fluids.

·         If there are no signs of dehydration, go on to Step 3 to maintain hydration by replacing continuing fluid losses.