Biochemical characteristics of isolates of
Cholera, also known as “blue death” is a potentially epidemic and life-threatening secretory diarrhea characterized by numerous voluminous watery stools, often accompanied by vomiting and resulting in hypovolemic shock and acidosis. The causative agent of this water-borne disease belongs to certain members of the species Vibrio cholerae (V. cholerae) which can also cause mild or unapparent infections. V. cholerae is a facultative anaerobic, Gram-negative bacilli, which possess the characteristic feature of darting motility under wet-mound preparations. Other members of the species may occasionally cause isolated outbreaks of milder diarrhea, whereas others—the vast majority—are free-living and not associated with disease. The emergence of new, virulent, drug-resistant strains of Vibrio is the main cause of protracted outbreaks leading to high fatality rates. The subsequent loss of fluid volume causes a drop in blood pressure and circulatory shock. If the patient remains untreated, they become progressively weaker, sometimes to the point of death, within 12–24 h of the onset of symptoms. The severity and fatality of the disease depend on the strain of Vibrio. The cholera toxin-producing (CT-producing) V. cholerae manifests the most fatal disease known as cholera gravis. Throughout most of the twentieth century, cholera was caused by V. cholerae of the O1 serogroup, and the disease was largely confined to Asia and Africa. The emergence of a pandemic in 1992 was caused by an unknown serogroup of V. cholerae (O139) wherein the targets were India and Bangladesh. The pathogenesis and virulence of the bacteria are due to an enterotoxin it produces—cholera toxin (CT). The mechanism of action of CT is discussed in this chapter at a later stage. Attempts have been made to produce vaccines through a number of trial-and-error methods, and still the possibility of an effective vaccine which gives a good prophylactic measure is under consideration. The identification of the bacteria as well as toxin detection is one of the main elements in the clinical microbiology field. The identification and confirmation of this epidemic disease commend from the morphological identification of Vibrio and end with serotyping and biotyping in addition to toxin detection by various means of assays—both in vitro and in vivo. This chapter will cover all of the mentioned areas of clinical microbiology with respect to cholera infection in addition to the recent outbreaks and epidemics which occurred across the globe.
- Vibrio cholerae
- Vibrio cholerae O139
- vibriocidal antibodies
- secretory IgA
Cholera, a water-borne infectious disease (also known as “blue death”), is characterized by life-threatening secretory diarrhea often accompanied by numerous voluminous watery stools and vomiting, eventually leading to hypovolemic shock and acidosis. The term “blue death” is given to the disease as it changes the skin of the infected individuals to blue discoloration due to severe dehydration [1, 2]. It is caused by certain members of the species
2. Clinical features
The symptoms of the cholera disease usually manifest 1–3 days after ingestion of contaminated food and/or water. In most cases, the symptoms range from mild to moderate, but in 20% of cases, severe life-threatening fatal conditions appear. The most severe manifestation of the disease, “cholera gravis,” affects only minority of the patients infected with CT-producing
Lactic acidosis and stool bicarbonate losses associated with dehydration are manifested by decrease of blood pH and plasma bicarbonate and an increased serum anion gap (mean of 20.2 mmol/l in a study). Despite potassium loss, untreated acidosis usually leads to high potassium levels in serum. In children, hypoglycemia associated with coma and convulsions may occur [4, 6].
2.2. Mechanism of pathogenesis
A better understanding of the source of infection is very much necessary for the management and effective prophylaxis of any diseases. In the case of cholera, contaminated water and feces of persons infected with
The mechanism of invasion begins as soon as the bacterium establishes itself in the bowel, following the colonization of the epithelial layer of the small intestine by penetrating the mucous. The penetration of the mucous is facilitated by mucolytic enzymes which help to destroy the mucous integrity, and also the long tail of the invading organism allows it to propel itself through the thick mucosal layer. The attachment
The CT that is made is comprised of six protein subunits: one A subunit and five copies of B subunits, generally denoted as AB5. B subunit is also known as the binding factor which binds to the GM1 ganglioside receptor of the epithelial cells of the small intestine. Once bound with the target cells, it forms a toxin complex which is then endocytosed by the cell. As soon as the process of endocytosis takes place, the enzymatic activation of A subunit occurs leading to increased adenylate cyclase activity, thereby increasing the concentration of cAMP to more than 120-folds. This in turns leads to increased permeability of the chloride channels subsequently mediating the efflux of more ATP-mediated chloride ions and secretion of mere H2O, Na+, K+ and HCO3− into the lumen of the intestine [4, 5, 7]. The increased absorption of water as well as electrolytes is responsible for the massive dehydration leading to the clinical symptoms of cholera. The brief mechanism of pathogenesis is depicted in Figure 1.
2.3. Differential diagnosis
In places where cholera disease is found epidemic or endemic, treatment should never be delayed due to pending laboratory investigations; laboratory confirmation is very essential for the prophylaxis as well as for treatment in epidemic-endemic areas.
As outlined above, definitive diagnosis is required for which the most crucial step is the isolation of
The key to therapy is adequate rehydration till the disease has come to its end course (usually 1–6 days in the absence of antibiotics). Rehydration can be accomplished by oral rehydration with oral rehydration solution (ORS) as in other cases of diarrhea and dysentery. In severe cases, intravenous infusion of fluid will be necessary. Antibiotics help to reduce the severity of illness and the duration of excretion of the organism. In children, hypoglycemia is treated with 25 or 50% glucose administered intravenously .
2.5. Intravenous rehydration
For adults, intravenous rehydration is done as fast as possible; 2 liters is administered the first 30 minutes. If the condition of the patient improves at this point, the rate of infusion can be lowered to 100 ml/kg of the body weight within the first 4 hours of therapy. Children should receive 30 ml of the intravenous fluid per kg of body weight during the first hour and an additional 40 ml/kg in the following 2 hours [10, 11]. Both adults and children should receive appropriate amount of ORS during the course of illness. The rate and frequency of intravenous infusion can be varied depending on the electrolyte loss from the stool of the patient. The World Health Organization (WHO) recommends Ringer’s lactate as the best rehydration solution. Isotonic saline corrects only hypovolemia, so potassium and glucose should be administered along with it for proper recovery from dehydration [12, 13].
2.6. Oral rehydration
Patients with moderate illness and dehydration can receive fluid replacement orally to compensate for the water and electrolyte depletion. In mild cases, the WHO recommends 50 ml of ORS/kg of body weight to be given in the initial 4 h. For moderate dehydration, the quantity should be increased to double, i.e., 100 ml/kg [11, 14]. Stool output should be continuously monitored depending on which the rehydration therapy is optimized. For children, who tend to have high fluid losses, it should be replaced 1:1 with ORS. The oral solution used must be the one recommended by the WHO . This solution consists of appropriate amount of electrolytes to balance the fluid and electrolyte losses due to dehydration. In addition, it also contains optimum quantity of glucose to facilitate the absorption of sodium and water.
The formula for the current WHO oral rehydration solution is 2.6 grams table salt (NaCl), 2.9 grams trisodium citrate dihydrate, 1.5 grams (0.053 oz) potassium chloride (KCl) and 13.5 grams anhydrous glucose (C6H12O6) per liter of fluid [1, 6, 12].
A homemade oral rehydration solution can be prepared when readymade sachet is not available. The composition of the ORS solution consists approximately of six level teaspoons of sugar and 1/2 a teaspoon of table salt in 1 liter of water. The molar ratio of sugar to salt should be 1:1, and the solution should not be hyperosmolar [9, 12].
2.7. Antimicrobial therapy
Treatment with antibiotics should start only after the initial rehydration therapy and correction of acidosis. Antibiotics play a secondary role in the treatment of cholera by shortening the length of cholera diarrhea as well as the excretion of vibrios. Though most of the
Cholera is an indication of the prevalence of unsanitary conditions especially with the growing populations in developing countries. It is a plight that about 3–5 million cases of cholera are reported each year with the advancements of health and medical professionals across the globe . When 52 countries reported 236,896 cases in 2006 with a fatality rate of 2.7%, a total of 589,854 cases were reported in 2011 from 58 countries . These numbers suggest cholera being a global disease threatening worldwide.
In developing countries, where the population is high, cholera is the main cause of epidemic diarrhea. Global pandemic outbreaks are also being reported from Asia, Africa and Latin America . Africa, being a country with limited medical facilities, has shown a considerable decrease in cholera outbreaks in the recent years. When 117,570 cases were reported in 2012, only 56,329 cases were reported in 2013 . This shows a remarkable sanitary improvement as well as prophylactic measures adopted by the country. During 2013, Mexico and other Central African countries reported an outbreak, while 2014 witnessed a cholera outbreak in South Sudan. In an outbreak in Cuba, a total of 185 cases were reported in 2012 . The WHO reports an average of 132,121 cholera cases in 2016 from 38 countries which includes 2420 fatality reports .
Considering the epidemiology of the different serotypes and biotypes of
4. Immune response and vaccines
Over 75 years of research has been done on the development of an effective vaccine against cholera. Since
4.1. Infection-derived immunity
The immunity induced by cholera infection is strain and serotype specific. Studies show that volunteers experimentally infected with classical biotype
4.2. Components of the immune system
It is likely that the primary immune response might be a main determinant of protection against cholera infection since it is essentially a disease of the small intestine. Moreover infants under breastfeeding are less likely to be affected due to the presence of the secretory antibodies present in the milk [19, 21]. Secretory IgA, IgM and IgG are found in the intestinal mucosa. These antibodies confer protective immunity in the presence and absence of complementary proteins. The actual protection is due to the secretory IgA and other intestinal antibodies, and it is the vibriocidal antibodies in the serum which serves as the marker for IgA antibodies to direct against the same antigens [12, 21].
The observation that natural infection confers long-lasting immunity has led to the development of cholera vaccines which can elicit protective immunity. The first attempts of vaccine development started in 1960, which focused on whole-cell vaccine preparation with parenteral administration. Ninety percent immunity was obtained, but it was not long-lasting as the antibody baseline titer waned in an year . An attempt has also been made by converting cholera toxin to toxoid in the presence of glutaraldehyde and formalin, but it had elicited a very low level of protection as toxoid is a poor antigen .
In recent years, the importance of intestinal immune response has paved a different direction in vaccine development, i.e., a shift from parenteral to oral vaccines. Parenteral administration of antigens elicits the stimulation of secretory antibodies (IgA, IgG, and IgM), but the antibodies have a short life span and a poor memory . In controversy to it, oral administration of antigens elicits immune response of a longer shelf life and good memory proving to be a good vaccine. There is not much contribution from serum antibody in cholera infection as
The FDA has approved a single-dose live oral vaccine for cholera called Vaxchora (lyophilized CVD 103-HgR) for the age group 18–64 years old . Though it is not recommended in the usual immunization category, it is administered for travelers who are moving to a region actively spread with the toxigenic vibrios. As per the current scenario, there is no cholera vaccine which provides 100% protection, and vaccination is not recommended as a prophylactic measure in preventing cholera by the WHO.
The vaccines under trial consist of live attenuated whole-cell vaccines and killed bacterial suspensions, both of which are administered orally.
5. Laboratory investigations
5.1. Isolation and biochemical identification of the bacteria
Based on the somatic O antigens, currently there are about 130 serogroups of
Overnight growth (18–24 hours) of
Kligler’s iron agar
Triple sugar iron agar
Glucose (acid production)
Glucose (gas production)
Sucrose (acid production)
K/A, no gas, no H2S
A/A, no gas, no H2S
5.2. Serologic identification
As discussed previously, there are more than 130 serogroups of
Isolates of O1 serogroup is further divided into three serotypes, namely, Ogawa, Inaba and Hikojima (rare). Strains that are positive with polyvalent O1 antisera are further tested for agglutination with Ogawa and Inaba antisera. In some instances, there may be strains that agglutinate very strongly and equally with both Ogawa and Inaba antisera. If ever such rare reactions are suspected, those strains should be referred to a reference laboratory for further confirmation and may be referred to as “possible serotype Hikojima” [9, 12].
|Serotype||Major O factors present||Agglutination in absorbed sera|
|Hikojima||A, B, C||+||+|
5.3. Tests to distinguish biotypes of
The typing of
|Voges-Proskauer (modified with 1% NaCl)|
|Zone around polymyxin B (50 U)|
|Agglutination of chick erythrocytes|
|Lysis by bacteriophage|
El Tor V
5.4. Toxin assays
The virulence of the O1 serogroup is marked by the production of CT toxin. The CT molecule comprises one A (active) subunit and five B (binding) subunits. The B subunit attaches to the GM1 ganglioside receptors on epithelial cells of the intestinal mucosa during the process of infection. After this process, cleavage occurs between the subunits so as to facilitate the entry of A component into the cell. The A1 component is responsible for the stimulation of adenylate cyclase which in turn produces cyclic AMP (cAMP) [3, 12]. Increased intracellular production of cAMP leads to disruption of electrolytes across the cell membrane leading to fluid secretion into the small intestine. When the fluid entering the intestine is greater than its reabsorption capability, it leads to diarrhea [12, 23]. CT toxin is similar to
Before testing for the toxin, the isolate should be confirmed as
220.127.116.11. Animal methods
The first cholera enterotoxin assay was the adult rabbit ileal loop method in the 1950s in which the enterotoxin was injected into the ligated segments of the intestine (ileal loops) of experimental animals such as rabbits, pigs, dogs and calves, and this caused accumulation of fluid. This mechanism is routinely used for the study of mechanism of CT, heat labile toxin of
Another model was developed in 1955, the infant rabbit infection model which was also used for the determination of different toxins. Here, a 7-day-old infant rabbit is infected with the test culture either by gastric intubation or by intraluminal injection. A positive assay results in watery diarrhea of the rabbit and eventual death due to dehydration. The small intestine is removed, and the loops are measured to quantify the excess fluid accumulation . The drawback of this procedure is the requirement of one animal per test sample.
The vascular permeability factor assay is a dermal test wherein the activity of CT or LT is determined by the neutralization of standardized amount of specific antisera against CT. Here 20–30 supernatants can be injected per rabbit. A cell-free supernatant is injected intradermally into the rabbit, following which Evans blue dye is injected intravenously. The increased capillary permeability mediated by CT leads to perfusion of the dye in the skin causing a blue coloration with localized induration at the site of injection. The diameter of discoloration and induration is measured with a negative control [1, 12].
18.104.22.168. Tissue culture methods
Tissue culture methods are widely used in laboratories nowadays for the toxin assay. It requires skilled professionals and equipment in the laboratory with existing tissue culture facilities. It is a very sensitive assay with high reproducibility. In addition to detecting toxins, these assays are also useful in detecting toxin-neutralizing antibodies.
The Chinese hamster ovary (CHO) and the Y1 mouse adrenal (Y1) cell lines are used for the assay of CT and LT, although Vero monkey kidney cells are also used. The toxin if present in the cell-free supernatant increases the intracellular production of cAMP in the cell lines. This can be made out by the morphological changes in the cell lines (Y1 cells round and CHO cells elongate) [7, 12].
As CT and LT are immunogenic to both humans and animals, many immunogenic assays have been developed to detect the presence of toxins in vitro. As discussed earlier, the GM1 ganglioside is the natural receptor for CT. In ELISA method, this GM1 ganglioside receptor is purified and is used for the preparation of ganglioside-capture enzyme-linked immunosorbent assay. The cell-free supernatant in which the toxicity is to be tested is added to the microtiter wells coated with GM1 ganglioside. CT antiserum is then added on the plate to detect the binding of the toxin with the bound receptor on the plate, followed by the addition of enzyme-conjugated antiglobulin antibody .
22.214.171.124. Latex agglutination
In this assay, highly purified specific anti-CT is bound to latex particles. A commercial version of kit is available in the market which allows easy detection of cholera toxins in the laboratory.
5.4.3. DNA-based assays
126.96.36.199. Polymerase chain reaction (PCR)
PCR is a molecular approach based on specific DNA sequences. In PCR, DNA polymerase enzyme is used to amplify certain specific sequences of DNA (amplicons) which can be detected on an agarose gel or using specific DNA probes . The toxigenicity of
|Assay||Sensitivity (per ml)||Type of assay||Specific target of assay||Sample tested|
|Rabbit ileal loop||30 ng||Bioassay||Stimulation of fluid accumulation||Culture supernatant|
|Infant rabbit assay||250–500 ng||Bioassay||Stimulation of fluid accumulation||Broth culture/supernatant|
|Rabbit skin test||0.1–3.5 ng||Bioassay||Permeability factor||Culture supernatant|
|Y1 mouse adrenal cells||10 pg||Bioassay||Accumulation of cAMP||Culture supernatant|
|10 pg||Bioassay||Accumulation of cAMP||Culture supernatant|
|GM1-ELISA||10 pg||Immune||B subunit||Culture supernatant|
|Coagglutination||50 ng||Immune||B subunit||Culture supernatant|
|Reverse passive latex agglutination||1–2 ng||Immune||B subunit||Culture supernatant|
|DNA probe||Detects Ctx gene||Genetic||Ctx gene||DNA (colony blot)|
6.1. Future perspectives of the chapter
The Centers for Disease Control and Prevention (CDC) is always one step ahead in investigating any cholera outbreaks across the globe. They have trained professionals and sophisticated laboratory protocols for identification of toxigenic vibrios and emergence of any new antigenically modified strain as the Haiti outbreak. The WHO in collaboration with the CDC also provides information to the public and the health officials regarding the necessity of proper sanitization and preventive control measures to overcome the dreaded disease. The antigenic variation of the