3/7/2015

Cholera: Overview

Vibrio
Dr Debasis Biswas

Epidemiology Microbiology
1854:
John
Snow
investigated
an
epidemic of cholera in
London .
identified water from
the Broad Street pump
as the likely source of
the
disease

removal of the pump

handle contained the
epidemic

Endemic in India
Till 1817
Ganga &
Brahmaputra
Basins
Periodic outbreaks

Vibrio cholerae.
Fecal-oral route.
Profuse secretory diarrhea.
Endemic, epidemic, or pandemic.
Asymptomatic Mild Severe cholera causing
dehydration and death within hours of onset.
Definitive diagnosis: Not a prerequisite for treatment.
Priority in management: Replacing lost f luid and
electrolytes and providing an antimicrobial agent when
indicated.

Epidemiology Microbiology
1883:

Robert
Koch
independently identified
V. cholerae during an
outbreak in Egypt.

The genus name refers to

the fact that the organism
appears to vibrate when
moving.

Cholera pandemics
Since 1817: 7 cholera pandemics
1817- 1923: First 6 pandemics V cholerae O1 of
classical biotype
All 6 pandemics originated from choleras endemic
reservoir in the Indian subcontinent.
1961- till date: 7th pandemic . V cholerae O1 of EI
Tor biotype
7th pandemic originated from the Celebes Islands,
Indonesia affected more countries and continents
than the previous 6 pandemics.
Environmental Resilience: El Tor > Classical
Virulence: Classical > El Tor
El Tor: Carriers +++; Endemicity: +++

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O139 (Bengal)

Biotypes & Serotypes

Emerged in the fall of 1992 form Madras Outbreaks

in Bangladesh and India in 1993.
11 countries in SE Asia have reported isolation of this
serogroup.

V. cholerae O1: Biotypes Classical & EI Tor

Clinically and epidemiologically indistinguishable from

disease caused by O1 strains.

Both biotypes are subdivided into 3 serotypes:

Ogawa O antigens a & b
Inaba O antigens a & c
Hikojima O antigens a, b & c

Some experts regard this as an 8th pandemic.

ElTor has regained its dominance since 1994

Test

Classical Vibrio El Tor Vibrio

Haemolysis

Voges Proskauer

Chick erythrocyte
agglutination
Polymyxin B Sensitivity

Group IV Phage
Susceptibility
EL Tor Phage V
Susceptibility

Pathogenesis

S o u r c e : H u m a n E xc r e m e n t
R e s e r v o i r s : H u m a n & Wa t e r
R o u t e o f i nf ec t i o n: Faeco - o r al
Ve h i c l e s : C o n t a m i n a t e d w a t e r, f o o d , f o m i t e s

Gastric Acidity: Not acid- resistant

Infectivity influenced by: inocculum size; gastrectomy; antacids; H2
blockers; PPI
ID with water: 103 -106 ; ID with food: 102 -104
Small Intestine: Secretion of Enterotoxin: AB5
B subunits bind to ganglioside
receptors on intestinal epithelium

A subunit: increase in cAMP levels

Pathogenesis
Faec o - o r al r o u te o f inf ec tio n:
C o n t a m i n a t e d w a t e r, f o o d , f o m i t e s
Gastric Acidity: Not acid- resistant
Infectivity influenced by: inocculum size; gastrectomy;
antacids; H2 blockers; PPI
Small Intestine: Secretion of Enterotoxin: A2B5
B subunits bind to ganglioside
receptors on intestinal epithelium

A subunit: increase in cAMP levels

cAMP: blocks abs. of Na & Cl by microvilli

Promotes secretion of Cl & water by crypt cells

cAMP: blocks absorption of Na+ & Cl- by microvilli

Promotes secretion of Cl- & water by crypt cells
VOLUMINOUS WATE RY DIARRH OEA
VOLUMINOUS WATE RY DIARRH OEA

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Pathogenesis: O139

Dehydration

Similar pathogenic mechanism except that it produces a

novel O139 lipopolysaccharide (LPS) and an
immunologically related O-antigen capsule.
These 2 features enhance its virulence and increase its
resistance to human serum in vitro and occasional
development of 0139 bacteremia.

Dehydration can develop with remarkable rapidity, within

hours after the onset of symptoms.
This contrasts with disease produce by infection from any
other enteropathogen.
Because the dehydration is isotonic, water loss is
proportional between 3 body compartment, intracellular,
intravascular, and interstitial.

Severity of Dehydration

Hypoglycemia

3-5% loss of normal body weight:

Excessive thirst
5-8% loss of normal body weight:
Postural hypotension
Tachycardia
Weakness, fatigue, dry mouth.
>10% loss of normal body weight:
Oliguria
Glassy or sunken eyes
Sunken fontanelles in infants
Weak, thready, or absent pulse
Wrinkled washerwoman skin
Somnolence
Coma.

2nd most common lethal complication of cholera in

children (After dehydration).
Result of: diminished food intake
exhaustion of glycogen stores
defective gluconeogenesis secondary to
insufficient stores of gluconeogenic
substrates in fat and muscle
May cause convulsions

Metabolic Acidosis

Hypokalemia

Bicarbonate loss in stools

Accumulation of lactate because of diminished
perfusion of peripheral tissues
Hyperphosphatemia.
Acidemia results when respiratory compensation is
unable to sustain a normal blood pH.

Potassium loss in the stool

Severe total body potassium depletion.
Mean K+ concentration maintained at 3.0 mmol/L.
(because of the existing acidosis)
Hypokalemia develops only after the acidosis is
corrected and intracellular hydrogen ions are
exchanged for extracellular potassium
May manifest as paralytic ileus.

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Hypocalcemia

Diagnosis

Rehydration therapy with bicarbonate-containing

fluids can also produce hypocalcaemia by decreasing
the proportion of serum calcium that is ionized.
Chvostek and Trousseau signs are often present, and
spontaneous tetanic contractions can occur..

WHO standard case definition:

o In an area where the disease is not known to be
present, a patient aged 5 years or older develops
severe dehydration or dies from acute watery
diarrhea.
o In an area with a noted cholera epidemic, a patient
aged 5 years or older develops acute watery
diarrhea, with or without vomiting.

Morphology

Gram Stain

Comma-shaped.
1-3 m (length) X 0.5-0.8 m (diameter).
Gram-negative.
Aerobic or facultatively anaerobic bacillus.
Polar flagellum: Flagellar H antigen.
Somatic O antigen . Serogroups: > 200
O1, O139: Cholera

Gram negative comma shaped bacteria

Electron Microscope images of Vibrio cholerae

Important biological properties

Darting Motility

Characteristic motility: Darting

V cholerae is not fastidious in nutritional requirements
for growth. However, it needs adequate buffering if
fermentable carbohydrate is present.
Viability is severely compromised if pH< 6, . Resulting
in autosterilization of the culture.
Many of the selective media used to differentiate enteric
pathogens do not support the growth of V cholerae.
Colonies are lactose-negative, like all other intestinal
pathogens, but sucrose-positive.
Oxidase- positive: Unlike other Enterobacteriaceae

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Immobilization with antisera

Culture Media

Specific Anti- Vibrio antisera can be used in

immobilization tests.
A positive immobilization test result (i.e. cessation
of motility of the organism) is produced only if the
antiserum is specific for the Vibrio type present

Transport Media: Contain NaCl

Venkatraman Ramakrishnan Medium (pH 8.6-8.8)
Cary- Blair Medium (pH 8.4): Also for Salmonella, Shigella

Ability to grow at a high pH or in presence of bile salts

Halotolerant (NaCl: 0.5-1% reqd for optimal growth)

Enrichment Media:
Alkaline peptone water (pH 8.5-9)
Monsurs taurocholate tellurite peptone water (pH 9.2)
Selective Media:
Thiosulfate-citrate-bile-sucrose-agar (pH 8.6)
Large, smooth, round yellow colonies
Monsurs Gelatin Taurocholate Trypticase Tellurite Agar
Small, translucent colonies with greyish black center
and turbid halo

Colony Characteristics

Cholera Red Reaction

Few drops of conc. Sulphuric Acid added to
peptone water culture

McConkey Agar: Pale Colonies

TCBS: Yellow Colonies

PCR

El Tor Variant

Polymerase chain reaction (PCR) tests for identifying V

cholerae have been developed. These have a high
degree sensitivity and specificity.

Emerged in 1992
Genetic backbone of the El Tor biotype
Produces cholera toxin formerly produced only by
classical strains.
Has replaced the original El Tor strain in several parts of
Asia and Africa.
Associated with more severe disease, higher CFRs

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Treatment

Cholera: Current Challenges

Mild to moderate dehydration

Oral rehydration solutions: Salts and glucose
Rice based oral rehydration salts
Low osmolarity solutions
Severe dehydration cases
Intravenous rehydration: Ringers lactate solution
Antibiotics are a part of the treatment of severe cholera
but are not needed for mild cases and are
contraindicated for prophylaxis.
Locally relevant Antibiotic sensitivity profile should
guide choice of antibiotics.

Increased frequency of large, protracted and often

uncontrolled cholera epidemics in Zimbabwe and
other African and Asian countries in the past few
years
Global warming: Rising sea levels, increases in water
temperature
Growing urbanization, growth in slums: strained
hygiene, water and sanitation systems.
Emergence of antibiotic- resistant strains
Emergence of El Tor variants: More transmissble,
more virulent

Oral Cholera vaccines

Dukoral

Killed Vaccine
Two Types:
Dukoral
Shanchol and mORCVAX: identical strains but
formulated by different manufacturers using different
methods.

Whole cells (WC) of V. cholerae O1 (Classical and El

Tor, Inaba and Ogawa: formalin and heat-killed
Plus
Recombinant Cholera Toxin B subunit (rBS).

Oral live attenuated single-dose vaccine (CVD 103-HgR)

is no longer produced

WC- rBS

To protect the toxin B subunit from being destroyed by

gastric acid, the vaccine must be given with a
bicarbonate buffer.

Injectable vaccine prepared from phenol-inactivated

strains:
Limited efficacy
Short duration of protection
Not recommended by WHO

Dukoral:
Dose schedule
Primary immunization:
For adults and children aged 6 years
2 oral doses 7 days apart (but < 6 weeks apart).
For children aged 2-5 years
3 doses 7 days apart (but <6 weeks apart)

3ml single-dose vials

+ Bicarbonate buffer (effervescent granules in sachets).
Mixed in 150ml of water for persons aged >5 years and
in 75ml of water for children aged 2-5 years.
Shelf life of 3 years at 2-8OC and 1 month at 37OC
Safe in pregnancy and in HIV/immunocompromised pts.

No food or drink for 1 hour before and after vaccination

Protection expected about 1 week after the last scheduled
dose.
Not licensed for children aged <2 years
Booster dose: If continued risk of infection
For adults and children aged 6 years
1 dose after 2 years
For children aged 2-5 years
1 dose after 6 months

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Dukoral: Immunogenicity

Shanchol/ mORCVAX

Both antibacterial and antitoxin antibodies

Mucosal IgA antibodies produced locally in the
intestines

Shanchol: India
mORCVAX: VietNam
Serogroups O1 and O139.

Cross- protection against ETEC

No Cholera Toxin B subunit

No protection from ETEC

Shanchoral: Dose schedule

Shanchol: Protective Efficacy

Bivalent

After 2 doses: 66% protection for at least 2 years (95%

CI, 46-79%) in endemic conditions
Similar results in children aged 1-5 years and adults.

Primary immunization:
For adults and children aged 1 year
2 oral doses 14 days apart (but < 6 weeks apart).
Booster dose: After 2 years

Advantage Shanchol

Herd Immunity

Easier operation in field conditions:

Does not require a buffer or water
Less storage space reqd

Confer considerable herd protection

Also for children too young to be vaccinated.
During the first year after vaccination

Cheaper (due to omission of the B subunit)

Efficacy: More effective and lasting protection
Both O1 & O139 strains

Vaccine coverage: >51%

Cholera incidence:
1.47/1000 placebo
recipients

Vaccine coverage: <28%

Cholera incidence:
7/1000 placebo
recipients

Matlab trial, Bangladesh

Vaccinating only 50% of the population: 93% reduction
in cholera incidence, taking into account both direct
protection and herd immunity

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WHO position on oral

cholera vaccines

Vibrio mimicus

Should be used in conjunction with other prevention

and control strategies (improving water and sanitation)
in endemic areas and should be considered in areas at
risk for outbreaks.

Mimics V. cholerae
Carries genes encoding Cholera toxin
Non- halophilic
Consumption of raw oysters
Severe Gastro-enteritis

Vibrio parahemolyticus

Vibrio alginolyticus

Halophilic: 6-8% NaCl concn.

Food poisoning: Seafood- Associated
Distributed in fishes & shellfish
Cytotoxin: Hemolysin
Explosive diarrhea: May be bloody
Kanagawa phenomenon: Clinical specimens
Hemolysis on Wagatsumas Agar
Sucrose- negative: Green colonies on TCBS

Bio-type 2 of V. parahemolyticus
Sucrose- fermenting
Swarming
Wound infection: Self- limiting
Mild cellulitis with sero-purulent exudate

Vibrio hollisae

Vibrio vulnificus

Contains gene sequences similar to hemolysin of V.

parahemolyticus
Raw seafood
Bacteremia & Diarrhea

Halophilic
Polysaccharide capsule: Anti-phagocytic
+ Toxins: Collagenses, Proteases,
3 distinct clinical syndromes
Fulminating Septicemia, followed by cutaneous lesions

with ecchymoses and bullae

Consumption of Raw shellfish
Invades Portal Venous System
Alcoholic Liver ds
Fatality rate: 50%
Cellulitis
Wound sustained in aquatic environment
Diarrhea
Consumption of Raw shellfish