1. Vibrio cholerae update
Dr.T.V.Rao MD
Dr.T.V.Rao MD

2. Cholera affects Millions
CHOLERA AFFECTS millions, in endemic areas and causes thousands of deaths especially during seasonal epidemics. Robert Koch, the famous microbiologist of Germany, discovered Vibrio cholerae, the causative organism. Because of its characteristic shape he originally referred to it as comma bacilli. Nearly 7-8 epidemics caused by the pathogen `Vibrio cholerae affected different parts of the world.
Dr.T.V.Rao MD

3. Modes of Transmission Water (infectious dose = 109)
Food (infectious dose = 103)
Person-to-person
Unless otherwise noted, the information presented in the notes section was taken from the website:
Sudden, large outbreaks are usually associated with water supply contamination. V. cholerae transmission has also been linked to drinking water drawn from shallow wells, rivers or streams, and even to bottled water and ice.
Food is the other important source of V. cholerae transmission. Seafood, especially raw or undercooked shellfish harvested from sewage-contaminated beds or environments where V. cholerae naturally occurs, has repeatedly been shown to be a source of V. cholerae infection.
V. cholerae grows well on moist, alkaline foods from which other competing organisms have been eliminated by cooking.
Fruits and vegetables grown in sewage and eaten without cooking or other decontamination are potential vehicles for cholera transmission.
Freezing foods or drinks does not prevent cholera transmission.
Person-to-person contact has not been shown to occur, but may, according to the WHO, still be a possible source of infection.
The 19th-century illustration depicting the spirit of death at a pump was taken from
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4. Discovery of Cholera Organisms
Cholera came to Florence in 1854 during the Asiatic Cholera Pandemic of Pacini became very interested in the disease.  Immediately following the death of cholera patients, he performed an autopsy and with his microscope, conducted histological examinations of the intestinal mucosa. During such studies, Pacini first discovered a comma-shaped bacillus which he described as a Vibrio. 
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5. Spread of Cholera Pandemics
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6. Filippo Pacini
Filippo Pacini, would gain prominence for his discovery of Vibrio cholera, but not until 82 years after his death, when the international committee on nomenclature in 1965 adopted Vibrio cholerae Pacini 1854 as the correct name of the cholera-causing organism.  Until then, many credited Robert Koch ( ) with this seminal discovery.
Dr.T.V.Rao MD

7. Robert Koch Isolates V.cholrae 1883
The German physician Robert Koch, like most of the scientific community, was unaware of Pacino's work at the University of Florence.  Yet both independently came to a similar conclusion.  Since Koch's findings eventually became accepted by his scientific peers, and were widely know in the popular press, he became the acknowledged discoverer of the cholera organism.  
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8. Cholerae Outbreaks
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9. Vibrio's in Nature
Vibrio's are among the most common bacteria in surface water worldwide.
They appear curved aerobic rods and are motile,possesing a polar flagellum
V.cholrae serogroups O1 and O139 cause cholera in humans, while other vibrio's may cause sepsis or enteritis
Dr.T.V.Rao MD

10. Vibrio cholerae
Epidemiology and spread of Cholera closely parallels the recognition of V.cholrae transmission in water and the development of sanitary system
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11. Morphology and Identification
V.cholrae is a comma shaped curved rod 2 – 4 µm long’
It is actively motile by means of polar flagellum.
On prolonged cultivation, vibrio's may become straight rods that resemble the gram-negative enteric bacteria.
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12. Vibrio Cholerae Vibrio spp. V. cholerae Non-O1 V. cholerae O1 Biotype
Classical
El Tor
Serotype
Ogawa
Inaba
Toxin
Toxigenic
Non-toxigenic
Other vibrios
Not all cholera is equal, this chart shows the different sub species. El Tor most commonly causes outbreaks.
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13. Vibrio cholerae O1 Salt resistant Heat and acid sensitive
El Tor biotype
Asymptomatic infections common
75% asymptomatic
18% mild diarrhea
1-5% severe-cholera gravis
Fast growing in food
Lengthy survival in environment
The cholera bacteria can survive for several months in water with plankton and algae helping it last even longer
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14. Vibrio cholerae O1 Infectious dose: 106 – 108 Incubation period
Varies with vehicle of transmission
Gastric acidity
Incubation period
1-3 days (½-5 days)
Infectious dose has to be very high, but due to growth rate, even small volumes of water may contain high enough inoculums, or an infectious amount of bacteria.
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15. Transmission-worldwide
Contaminated water
Contaminated food
Raw or undercooked seafood
Rice, cereals, gruels left at ambient temperature
Person to person transmission not common
Fecal-oral transmission is possible
Person-to-person transmission is not typical– however, proper isolation and care of patients is important in addressing outbreaks
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16. Known Virulence Factors
Integrons
Toxins CT
HA Protease
RTX Toxin
ACE and Zot
Adherence/Adhesins
Accessory Colonization Factors (ACF)
OmpU & other Omp Proteins - outer membrane proteins
Mannose-fucose-resistant cell hemagglutinin & Mannose sensitivev hemagglutinin (Faruque, 2002)
Toxin Co-regulated Pilus (TCP)
The information presented on the slide was derived from information reviewed by (Cotter, Peggy Cotter & Victor DiRita 2000), unless otherwise noted:
Notes about this slide:
Integrons - allow V. cholerae to acquire genes from other organisms and convert them into functional genes (Faruque, 1998)
Toxins
CT protein - cholera toxin. It is the toxin responsible for inducing diarrhea in patients with cholera. It will be discussed in more detail later.
Zot - the zot gene product is homologous to a family of proteins which includes the gene I product of M13 and the corresponding gene I products of several other filamentous bacteriophage of E. coli, P. aeruginosa and Zanthomonas. The gene I product is an inner membrane protein required for the assembly of the filamentous phages. The previously described zona occludens molecule is probably not directly associated with the V. cholerae gene product, but is likely involved in the morphogenesis of CTX (Faruque, 1998).
Adherence Factors/Adhesins
ACF - accessory colonization factor. It may be involved in signaling between the chemotaxis motility system and the pilus assembly system.
TCP - I will be discussing the role of this virulence factor in detail later in the presentation.
ompU - outer membrane protein U, may be an adhesin, although the role of the protein has not been definitively assigned.
Image from:
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17. Enrichment Medium
Enrichment of the fecal specimens are done on Alkaline peptone water
Venkataraman Ramakrishan Meidum is simple medium can be used as transport medium
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18. Culture
V.cholrae produces convex, smooth, round colonies that are opaque and granular in transmitted light;
Grow well at 370c on many defined media.
Vibrio's grow at a very high pH ( 8-5 – 9-5 ) and are rapidly killed by acid
In resource poor laboratories MacConkey agar can be used.
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19. Selective Medium - TCBS
V.cholrae grows well on Thiosulphate citrate bile sucrose (TCBS ) agar, on which it produces yellow colonies that are readily visible against the dark green background of the agar.
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20. Growth Characteristics
V.cholerae ferments sucrose and mannose but not arabinose
A positive Oxidase test is key step in preliminary identification of V.cholerae and other Vibrio's
Vibrio species are susceptible to compound 0/129 and differentiates from Aeromonas
Vibrio's usually grow on medium containing 6% Nacl
Halophilic vibrios need and grow in the presence of > 6% Nacl
On Blood agar Vibrios show hemodigesion
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21. Antigenic Structure and Biological Classification
Many Vibrio's share a single healable H antigen. Antibodies to H antigen are probably not involved in the protection of susceptible hosts.
V.cholrae has O lipopolysaccharide that confer serologic specificity
There are at least 139 O antigen groups
V.cholrae strains of O group 1 and O group 139 cause classic cholera.
Occasionally non 01/non 0139 V.cholrae cause Cholera like disease
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22. V.cholrae - typing
V.cholrae 01 has determinants that make possible futher typing
Serotypes are
Ogawa, Inaba and
Hikojima.
Epidemic V.cholerae is biotyped into
1 Classic
2 El Tor
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23. El Tor Vibrio's
The El Tor vibrios produce a Hemolysin and positive results with Voges- Proskauer test
Resistant to Polymyxin B
Molecular techniques can also be used to type V.cholrae
Tests can be done reference laboratories and technically demanding
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24. New Epidemic of V. cholrae 0139
The epidemic of cholera caused by V cholerae 0139 has affected at least 1 1 countries in southern Asia. V cholerae 0139 produces severe watery diarrhea and dehydration that is indistinguishable from the illness caused by V cholerae 01, and appears to be closely related to V cholerae 01 biotype El Tor strains. Specific totals for numbers of V cholerae 0139 cases are unknown because affected countries do not report infections caused by 01 and 0139 separately; however, >100,000 cases of cholera caused by V cholerae 0139 may have occurred.
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25. V.cholrae
V.cholrae 0139 is very similar to V.cholrae 01 El Tor biotype
V.cholrae 0139 do not produce 01 type lipopolysaccharide and does not have all the genes necessary to make this antigen
V.cholrae 0139 make a polysaccharide capsule like other non 01 V.cholrae strains while V.cholrae 01 does not make a capsule
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26. V.cholrae 0139
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27. V.cholrae Epidemic
The epidemic of cholera caused by V cholerae 0139 has affected at least 1 1 countries in southern Asia. V cholerae 0139 produces severe watery diarrhoea and dehydration that is indistinguishable from the illness caused by V cholerae 01 and appears to be closely related to V cholerae 01 biotype El Tor strains.
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28. Vibrio cholerae - Enterotoxin
V.cholrae produce heat labile enterotoxin with a Moll wt. of about 84,000 consisting of sub units A ( MW 28,000 ) and B
Ganglioside GM1 serves as a mucosal receptor for subunit B, which promotes entry of subunit A into the cell
Activation of subunit A1 yields increased levels of intracellular cAMP and results in prolonged hyper secretion of water and electrolytes
There is increased sodium dependent chloride secretion, and absorption of sodium and chloride is inhibited
The genes for V.cholrae Enterotoxin are on the bacterial chromosome
Cholerae Enterotoxin is antigenically related to LT of Escherichia
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29. Mechanism of Action of Cholera Toxin
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30. Mechanism of Action of Cholera Toxin1 2
Mechanism of Action of Cholera Toxin 3 4
NOTE: In step #4, uptake of Na+ and Cl- from the lumen is also blocked.
HCO3- = bicarbonate which provides buffering capacity.
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31. Pathology and pathogenesis
V.cholrae is pathogenic to humans.
When bacteria are consumed with food few organism as much as 102 – 104 organisms are adequate to cause an attack because of the buffering capacity of the food
Any medication or conditions that decreases stomach acidity makes a person more susceptible to infection with V.cholrae
But a person with normal gastric acidity has to consume 1010 or more V.cholrae are when ingested with water
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32. Clinical events in Cholera
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33. Pathology Cholera is not an invasive infection
Organisms do not reach blood, only act locally
Virulent V.cholrae organism attach to the microvillus of the brush border of epithelial cells
They multiply and liberate cholera toxin and perhaps Mucinase and Endotoxin.
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34. Clinical manifestations
Diarrhea occurs as much as 20 – 30 Liters/Day fluids are lost.
Results in dehydration
Shock
Acidosis
Can lead to death.
About 60% of infections are caused with classic V.cholrae and are asymptomatic, about 75% of infections are caused by El Tor biotype
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35. Clinical features
The incubation period is 1 – 4 days for person who develop symptoms, depends on the size of the inoculums ingested
Manifest with
Nausea , vomiting,
profuse diarrhea, and abdominal cramps
Rice water stool characteristic of cholera
loss of fluid leads to profound dehydration
Circulatory collapse and anuria.
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36. Presentation- Rice water diarrheal
This slide shows a cup of the typical “rice water” stools, with flecks of mucus which have settled to the bottom. They are inoffensive, with a faint fishy odor. They are isotonic with plasma, with high levels of sodium, potassium and bicarbonate. They also contain extraordinary quantities of V. cholera 01 organisms.
Dr.T.V.Rao MD

37. Stool Examination Stool specimen appear as Rice water
On Microscopy contain Mucus, epithelial cells and large number of Vibrio's.
Milder cases difficult to differentiate from other diarrheal diseases.
El Tor vibrio's cause milder disease than classic biotypes.
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38. Specimen Collections
Stool specimens are collected in acute stage of the disease. before the antibiotics are administered.
Simple collection of stool in a wide Mouthed container is safe and hygienic.
Specimens should not be collected from bed pans.
Vomitus not advised.
Dr.T.V.Rao MD

39. Diagnosis Stool culture: Toxigenic Vibrio cholerae O1
Use Cary Blair Transport media if available
Viable for many days at room temperature
Use TCBS media for culture
Use V. cholerae serogroup O1 antisera
Confirm presence of cholera toxin
Cholera Rapid Test Dipsticks
Pass around cholera rapid test dipsticks
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40. Laboratory Diagnosis Mucus flecks from stool are cultured.
Smears are not useful for diagnosis.
Dark field microscopy shows rapidly motile vibrio's.
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41. Laboratory Diagnosis Culture
Growth is rapid on Blood agar,
On TCBS medium typical colonies can be picked in 18 hours.
The stool specimens can be transported in Venkataraman Ramakrishnan medium
Alkaline peptone water is ideal enrichment medium
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42. Bio Chemical Reactions
V.cholrae (El Tor)
+ve
-ve
V.cholrae( Classical )
Hemolysis ve
Voges -proskauer test -ve
Polymyxin sensitivity ve
Group IV phage
Susceptibility ve
Chick erythrocyte
Agglutination ve
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43. Confirmatory Tests for V.cholrae
V.cholrae organisms are further identified by slide agglutination tests using anti -0 group 1 or group 139 Antisera and by Biochemical reactions
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44. Immunity in Cholerae
Gastric acid produces some protection against cholera vibrios.
An attack of cholera if followed by immunity to reinfection but the duration and degree of immunity are not known.
In experimental animals specific IgA antibodies occur in the lumen of intestine
Vibriocidal antibodies in the serum titer > 1:20 have been associated with protection against colonization and disease
The presence of antitoxin antibodies have not been asociated with protection
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45. When you suspect Cholera
The diagnosis of cholera should be considered in patients with watery diarrheal who have recently (i.e., within 7 days) returned from cholera-affected countries. Patients with suspected cholera should be reported immediately to local and state health departments.
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46. Treatment Treat all cases / suspect cases promptly
Assess degree of dehydration
Determine if rehydration should be oral or IV
Don’t wait for laboratory confirmation to treat
Death rates from severe cholera can be decreased from ~50% to <1%
What about antibiotic use?
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47. Treatment
The most important part of therapy consists of correction water and electrolyte imbalance to correct severe dehydration and salt depletion.
Oral Tetracycline tends to reduce stool output in cholera and shortens the period of excretion of vibrio's
In some endemic areas tetracycline resistance has emerged the genes are carried by transmissible plasmids
Dr.T.V.Rao MD

48. Vaccines for Cholera
The licensed parenteral cholera vaccine provides only limited and brief protection against V cholerae 01, may not provide any protection against V cholerae 0139, and has a high cost-benefit ratio; therefore, the vaccine is not recommended for travellers. New oral cholera vaccines are being developed.
Dr.T.V.Rao MD

49. Newer Vaccines
New oral cholera vaccines are being developed and provide more reliable protection, although still at a high cost per case averted. None of these vaccines have attained the combination of high efficacy, long duration of protection, simplicity of administration, and low cost necessary to make mass vaccination feasible in cholera-affected countries.
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50. CDC – On Vaccination for Cholerae
Cholera vaccine is no longer required, nor recommended for the vast majority of travellers by the Centres for Disease Control and Prevention (CDC).
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51. Epidemiology of Cholerae
Six Pandemics of Cholera occurred between 1817 – 1923.
Most likely V.cholrae 01 of Classical type contributed to pandemics.
All pandemics originated in Indian continent.
The seventh pandemic originated in Celebes Islands in Indonesia on 1961.
Spread far and wide
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52. 8th Pandemic ? Spread of 0-139
Several identify that onset of is considered as 8th pandemic started in India.
Cholerae is spread by contact with persons in early or even mild illness.
By contaminated water, food, flies
Only 1 -5% of exposed will get effected
Carrier stage seldom exceeds 3- 4 weeks.
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53. Control of Cholerae Excreta disinfected All contacts to be followed up
Needs improvement of Sanitation associated with water treatment and food.
Patients infected preferably isolated .
Excreta disinfected
All contacts to be followed up
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54. Use of Vaccine and Chemoprophylaxis
Chemoprophylaxis with antibiotics is effective.
Repated injection of vaccine containing either Lipopolysaccharides extracted from Vibrio's or dense Vibrio's suspension can offer limited prevention to heavily exposed persons.
Vaccines not useful in Epidemic controls
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55. Prevention of Cholera
Although cholera can be life-threatening, it is easily prevented and treated. In the United States, because of advanced water and sanitation systems, cholera is not a major threat; however, everyone, especially travellers, should be aware of how the disease is transmitted and what can be done to prevent it.
Several regions in the Developing countries continue to be endemic locations.
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56. Cholera vaccines and vaccination
Two types of oral cholera vaccines are available: (i) Dukoral and (ii) Shanchol and mORCVAX. The latter two are identical vaccines in terms of strains but formulated by different manufacturers using different methods
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57. Characteristics of Dukoral, Shanchol and mORCVAX
The available oral cholera vaccines are safe29 and provide sustained protection of >50% that lasts for 2 years in endemic populations.30 Shanchol and mORCVAX have demonstrated longer term protection in children aged <5 years and do not require booster doses every 6 months.
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58. Potential use of oral cholera vaccines
Use of oral cholera vaccines in emergency situations is accepted but remains a challenge. To date, there is no specific indication for use of oral cholera vaccines in endemic situations, and intervention studies are being performed to prove their effectiveness as a public health tool.
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59. Other Bacteria resembling Vibrio cholerae
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60. Characteristics of Aeromonas and Plesiomonas Gastroenteritis
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61. Virulence Factors Associated with Non-cholerae Vibrio's
(Kanagawa positive)
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62. Characteristics of Aeromonas and Plesiomonas Gastroenteritis
Epidemiological Features
Aero monas
Plesiomonas
Natural Habitat
Source of Infection
Fresh or brackish water
Contaminated water or food
Fresh or brackish water Contaminated water or food
Clinical Features
Diarrhea
Vomiting
Abdominal Cramps
Fever
Blood/WBCs in Stool
Present
Absent
Pathogenesis
Enterotoxin (??)
Invasiveness
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63. Wish to Know More About Cholerae
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64. Programme created by Dr. T. V
Programme created by Dr.T.V.Rao MD for Medical and Health care workers in the Developing World
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65. Reference: www.Slideshare.com
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