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Vibrio cholerae
updateDr.T.V.Rao MD
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Cholera affects Millions
CHOLERA AFFECTS millions, in endemic areasand causes thousands of deaths especiallyduring seasonal epidemics. Robert Koch, the
famous microbiologist of Germany, discoveredVibrio cholerae, the causative organism.Because of its characteristic shape heoriginally referred to it as comma bacilli.
Nearly 7-8 epidemics caused by the pathogen`Vibrio cholerae affected different parts of theworld.
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Modes of Transmission
Water (infectiousdose = 109)
Food (infectiousdose = 103)
Person-to-person
The 19th-century illustration depicting the spirit of death at a pump was taken from
http://news.nationalgeographic.com/news/2004/06/0614_040614_tvcholera.htmlDr.T.V.Rao MD 3
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Discovery of Cholera Organisms
Cholera came to Florence in 1854 duringthe Asiatic Cholera Pandemic of 1846-63.Pacini became very interested in the
disease. Immediately following the deathof cholera patients, he performed anautopsy and with his microscope,conducted histological examinations of
the intestinal mucosa. During suchstudies, Pacini first discovered a comma-shaped bacillus which he described as aVibrio.
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Spread of Cholera Pandemics
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Filippo Pacini - 1854
Filippo Pacini, would gain
prominence for his
discovery ofVibrio cholera,
but not until 82 years after
his death, when theinternational committee on
nomenclature in 1965
adopted Vibrio cholerae
Pacini 1854 as the correctname of the cholera-causing
organism. Until then, many
credited Robert Koch (1843-
1910) with this seminal
discovery.Dr.T.V.Rao MD 6
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Robert Koch Isolates V.cholrae1883
The German physicianRobert Koch, like most ofthe scientific community,was unaware of Pacino'swork at the University of
Florence. Yet bothindependently came to asimilar conclusion. SinceKoch's findings eventuallybecame accepted by his
scientific peers, and werewidely know in the popularpress, he became theacknowledged discoverer ofthe cholera organism.
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Cholerae Outbreaks
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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
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Vibrio cholerae
Epidemiology and spread of
Cholera closely parallels therecognition of V.cholrae
transmission in water and thedevelopment of sanitary system
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Morphology and Identification
V.cholrae is a commashaped curved rod 2 4m long
It is actively motile bymeans of polarflagellum.
On prolonged
cultivation, vibrio's maybecome straight rodsthat resemble the gram-negative entericbacteria.
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Vibrio Cholerae
Vibrio spp.
V. cholerae
V. choleraeNon-O1
V. cholerae O1
BiotypeClassicalEl Tor
SerotypeOgawaInaba
ToxinToxigenicNon-toxigenic
Other vibrios
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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
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Vibrio cholerae O1
Infectious dose:
106 108
Varies with vehicleof transmission
Gastric acidity
Incubation period 1-3 days (-5
days)
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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
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Known Virulence Factors
Integrons
Toxins CT
HA Protease
RTX Toxin ACE and Zot
Adherence/Adhesins Accessory Colonization Factors (ACF)
OmpU & other Omp Proteins - outer membraneproteins
Mannose-fucose-resistant cell hemagglutinin &Mannose sensitivev hemagglutinin (Faruque,2002)
Toxin Co-regulated Pilus (TCP)Image from: http://www.amnh.org/nationalcenter/infection/01_mic/01a_bac.htmlDr.T.V.Rao MD 16
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Enrichment Medium
Enrichment of thefecal specimens are
done on Alkalinepeptone water
VenkataramanRamakrishan Meidum
is simple medium canbe used as transportmedium
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Culture V.cholrae produces convex,
smooth, round colonies thatare opaque and granular intransmitted light;
Grow well at 370c on manydefined media.
Vibrio's grow at a very highpH ( 8-5 9-5 ) and are
rapidly killed by acid In resource poor
laboratories MacConkeyagar can be used.
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Selective Medium - TCBS
V.cholrae grows well
on Thiosulphate
citrate bile sucrose(TCBS ) agar, on
which it produces
yellow colonies thatare readily visible
against the dark
green background ofthe a ar.Dr.T.V.Rao MD 19
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Growth Characteristics V.cholerae ferments sucrose and
mannose but not arabinose
A positive Oxidase test is keystep in preliminary identificationof V.cholerae and other Vibrio's
Vibrio species are susceptible tocompound 0/129 anddifferentiates from Aeromonas
Vibrio's usually grow on mediumcontaining 6% Nacl
Halophilic vibrios need and grow
in the presence of > 6% Nacl On Blood agar Vibrios show
hemodigesion
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Antigenic Structure and
Biological Classification
Many Vibrio's share a single healable H antigen.Antibodies to H antigen are probably not involved inthe protection of susceptible hosts.
V.cholrae has O lipopolysaccharide that conferserologic 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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V.cholrae - typing
V.cholrae 01 hasdeterminants that makepossible futher typing
Serotypes areOgawa, Inaba and
Hikojima.
Epidemic V.cholerae is
biotyped into1 Classic
2 El Tor
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El Tor Vibrio's
The El Tor vibrios producea Hemolysin and positiveresults with Voges-Proskauer test
Resistant to Polymyxin B
Molecular techniques canalso be used to typeV.cholrae
Tests can be donereference laboratoriesand technicallydemanding
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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. Vcholerae 0139 produces severe watery diarrhea anddehydration that is indistinguishable from the illness
caused by V cholerae 01, and appears to be closelyrelated to V cholerae 01 biotype El Tor strains.Specific totals for numbers of V cholerae 0139 casesare unknown because affected countries do not
report infections caused by 01 and 0139 separately;however, >100,000 cases of cholera caused by Vcholerae 0139 may have occurred.
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V.cholrae- 0-139
V.cholrae 0139 is verysimilar to V.cholrae 01 ElTor biotype
V.cholrae 0139 do not
produce 01 typelipopolysaccharide anddoes not have all the genesnecessary to make thisantigen
V.cholrae 0139 make apolysaccharide capsule likeother non 01 V.cholraestrains while V.cholrae 01does not make a capsule
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V.cholrae 0139
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V.cholrae 0139
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 01and appears to be closely related to V
cholerae 01 biotype El Tor strains.
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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 intracellularcAMP and results in prolonged hyper secretion of water andelectrolytes
There is increased sodium dependent chloride secretion, andabsorption of sodium and chloride is inhibited
The genes for V.cholrae Enterotoxin are on the bacterialchromosome
Cholerae Enterotoxin is antigenically related to LT ofEscherichia
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Mechanism of Action of Cholera Toxin
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Mechanism of Action of Cholera Toxin
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1 2
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Mechanism
of Action of
Cholera
Toxin
1
43
2
NOTE: In step #4,uptake of Na+ and Cl-
from the lumen is alsoblocked.
HCO3- = bicarbonate
which provides
buffering capacity. Dr.T.V.Rao MD 30
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Pathology and pathogenesis
V.cholrae is pathogenic to humans.
When bacteria are consumed with food feworganism as much as 102 104 organisms areadequate to cause an attack because of the bufferingcapacity of the food
Any medication or conditions that decreasesstomach acidity makes a person more susceptible toinfection with V.cholrae
But a person with normal gastric acidity has toconsume 1010 or more V.cholrae are when ingestedwith water
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Clinical events inCholera
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Pathology
Cholera is not an invasive infection
Organisms do not reach blood, only actlocally
Virulent V.cholrae organism attach to themicrovillus of the brush border ofepithelial cells
They multiply and liberate cholera toxinand perhaps Mucinase and Endotoxin.
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Clinical manifestations
Diarrhea occurs as much as20 30 Liters/Day fluids arelost.
Results in dehydration
Shock
Acidosis
Can lead to death.
About 60% of infections arecaused with classicV.cholrae and areasymptomatic, about 75%of infections are caused by
El Tor biotype Dr.T.V.Rao MD 35
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Clinical features
The incubation period is 1 4 days for personwho develop symptoms, depends on the sizeof the inoculums ingested
Manifest withNausea , vomiting,
profuse diarrhea, and abdominal cramps
Rice water stool characteristic of choleraloss of fluid leads to profound dehydration
Circulatory collapse and anuria.
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Presentation- Rice water diarrheal
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Stool Examination
Stool specimen appear asRice water
On Microscopy contain
Mucus, epithelial cells andlarge number of Vibrio's.
Milder cases difficult todifferentiate from otherdiarrheal diseases.
El Tor vibrio's cause milderdisease than classicbiotypes.
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Specimen Collections
Stool specimens arecollected in acute stageof the disease. before
the antibiotics areadministered.
Simple collection ofstool in a wideMouthed container issafe and hygienic.
Specimens should notbe collected from bedpans.
Vomitus not advised. Dr.T.V.Rao MD 39
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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
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Laboratory Diagnosis
Mucus flecks from
stool are cultured.
Smears are not
useful for diagnosis.
Dark field
microscopy shows
rapidly motilevibrio's.
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Laboratory Diagnosis
Culture
Growth is rapid onBlood agar,
On TCBS mediumtypical colonies can be
picked in 18 hours. The stool specimens
can be transported inVenkataraman
Ramakrishnan
medium Alkaline peptone water
is ideal enrichmentmedium
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Bio Chemical Reactions
V.cholrae( Classical )
Hemolysis -ve
Voges -proskauer test -ve
Polymyxin sensitivity +ve
Group IV phage
Susceptibility +ve
Chick erythrocyte
Agglutination -ve
V.cholrae (El Tor)
+ve
+ve
-ve
-ve
+ve
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Confirmatory Tests for V.cholrae V.cholrae organisms
are further identified
by slideagglutination tests
using anti -0 group 1
or group 139Antisera and by
Biochemical
reactions Dr.T.V.Rao MD 44
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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 immunityare not known.
In experimental animals specific IgA antibodies occurin the lumen of intestine
Vibriocidal antibodies in the serum titer > 1:20 havebeen associated with protection against colonizationand disease
The presence of antitoxin antibodies have not beenasociated with protection
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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 bereported immediately to local and
state health departments.Dr.T.V.Rao MD 46
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Treatment
Treat all cases / suspect cases promptly
Assess degree of dehydration
Determine if rehydration should be oralor IV
Dont wait for laboratory confirmation to
treat
Death rates from severe cholera can be
decreased from ~50% to
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Treatment The most important part of
therapy consists ofcorrection water andelectrolyte imbalance tocorrect severe dehydration
and salt depletion. Oral Tetracycline tends to
reduce stool output incholera and shortens theperiod of excretion of
vibrio's In some endemic areastetracycline resistance hasemerged the genes arecarried by transmissibleplasmids
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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.
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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 haveattained the combination of high efficacy, long
duration of protection, simplicity of
administration, and low cost necessary tomake mass vaccination feasible in cholera-
affected countries.
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CDC On Vaccination for CholeraeCholera vaccine is no longerrequired, nor recommendedfor the vast majority oftravellers by the Centresfor Disease Control andPrevention (CDC).
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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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8th P d i ?
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8th Pandemic ?
Spread of 0-139
Several identify that onset of 0-139 is considered as
8th pandemic started in India.
Cholerae is spread by contact with persons in early oreven 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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Control of Cholerae
Needs improvement of
Sanitation associated
with water treatment
and food.
Patients infected
preferably isolated .
Excreta disinfected
All contacts to be
followed upDr.T.V.Rao MD 54
Use of Vaccine and
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Use of Vaccine andChemoprophylaxis
Chemoprophylaxis with antibiotics is
effective.
Repated injection of vaccine containing
either Lipopolysaccharides extracted
from Vibrio's or dense Vibrio'ssuspension can offer limited prevention
to heavily exposed persons.
Vaccines not useful in Epidemic controlsDr.T.V.Rao MD 55
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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 majorthreat; however, everyone, especially
travellers, should be aware of how the disease
is transmitted and what can be done toprevent it.
Several regions in the Developing countries
continue to be endemic locations.Dr.T.V.Rao MD 56
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Cholera vaccines and vaccination
Two types of oral
cholera vaccines are
available: (i) Dukoral
and (ii) Shanchol andmORCVAX. The latter
two are identical
vaccines in terms of
strains but formulatedby different
manufacturers using
different methodsDr.T.V.Rao MD 57
Ch t i ti f D k l Sh h l
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Characteristics of Dukoral, Shancholand 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
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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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Other Bacteria resembling Vibrio
cholerae
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Characteristics of Aeromonas and
Plesiomonas Gastroenteritis
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Virulence Factors Associated with
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Virulence Factors Associated with
Non-cholerae Vibrio's
(Kanagawa positive)
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Characteristics of Aeromonas and
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EpidemiologicalFeatures Aero monas Plesiomonas
Natural Habitat
Source ofInfection
Fresh or brackishwater
Contaminated water
or food
Fresh or brackishwater Contaminated
water or food
ClinicalFeatures
Diarrhea
VomitingAbdominal
Cramps
Fever
Blood/WBCs in
Stool
Present
PresentPresent
Absent
Absent
Present
PresentPresent
Absent
Present
Characteristics of Aeromonas and
Plesiomonas Gastroenteritis
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Wish to Know More About
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Wish to Know More About
Cholerae
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Programme created by Dr.T.V.Rao MD
for Medical and Health care workers in
the Developing World
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