CHOLERA

Presenter: Dr. Sohani Bajracharya

Moderator: Prof. Dr. Basudha Khanal
Date: 26th February 2018
Introduction
• Cholera is an acute, diarrheal illness caused by infection of the intestine
with Vibrio cholerae, serogroup O1 or O139
• 3-5 million cases and over 100,000 deaths occur each year
• Infection is often mild or without symptoms, but can sometimes be
severe.
• Approximately, one in 10 infected persons will have severe disease
characterized by profuse watery diarrhea, vomiting and leg cramps
• In these people, rapid loss of body fluids leads to dehydration and shock
• Without treatment, death can occur within hours
Discovery of the causative agent

John Snow( 1849)- Filippo Pacini (1854) -

Robert Koch (1886)-
traced the source of first described and named
isolated and named
cholera outbreak ‘Vibrio’ – characteristic
Komma bacillus
vibratory motility
Epidemiology
• Cholera is native to the delta region of Ganga, Brahmaputra in West Bengal and Bangladesh
Pandemics Year Areas affected Causative agent
1st 1816-1826 India, China, Indonesia V. cholerae 01
Classical
2nd 1829-1851 Russia, Hungary, Germany, UK, France, North America
3rd 1852-1860 Russia, Indonesia, China,Japan, Philippines, Korea, UK,
Spain
4th 1863-1875 delta region of Ganges,Russia, Europe, Africa, North
America
5th 1881-1896 Europe, America, Russia, Spain, Japan, Egypt
6th 1899-1923 Philippines, India, Russia
7th 1961-1975 Indonesia, Bangladesh, Africa, Russia V. cholerae 01
EL Tor

• 1992-A new strain of cholera, Vibrio cholerae O139 Bengal, emerged and caused outbreaks
in Bangladesh and India.
Current Situation
• Cholera is a notifiable disease
• Often under reported, hence true incidence not known
• More than 3 million cases of cholera occur yearly (of which only 2
lakh cholera cases are reported to WHO), resulting in more than 1
lakh deaths annually ( of which <5000 are reported to WHO)
• Several epidemics have been reported such as from
Zimbabwe Seirra
Haiti(2010)
(2009) Leone(2012)

multiple
countries of Yemen(2015)
Africa (2014)
 Cholera in Nepal 2009
• Largest outbreak in Jajarkot , >30000 affected, >500 deaths

• Nepalgunj 200 affected, 6 deaths

• First epidemic recorded in 1823 2010

• Series of epidemics in Kathmandu • Saptari 111 affected, 2 deaths

2011
Valley in 1831,1843,1856,1862 and
1887 • three places affected Kathmandu, Doti, Bajhang, 13 deaths
2012
• Studies indicated that V. cholerae 01
• outbreak in Rautahat, >600 affected, 2 deaths
El Tor Ogawa is endemic in Nepal 2014

• Most cases reported during the • 82 confirmed cases reported , no deaths

rainy, or monsoon season 2015

• 110 cases, no deaths

2016

• 1 case reported, not verified by EDCD

2017
 Vibrio

• Curved, gram negative bacilli

• actively motile by a single polar flagellum
• fermentative, aerobic , oxidase positive , non sporing , non capsulated
• Habitat: found worldwide, coastal salt water and brackish estuaries
• 35 Vibrio species, 12 associated with human infections
• Vibrio cholerae : devastating diarrheal disease, responsible for global
pandemics and several epidemics
Gardner and Venkatraman Classification
Vibrio

Vibrio cholerae Other Vibrio spp.

O1 O139 Non
Serogroups
O1/O139

Biotypes Classical El Tor

Serotypes Ogawa Inaba Hikojima

Pathogenesis
• Cholera is a toxin mediated disease
• O1 and O139 are capable of producing cholera toxin
• Mode of transmission: ingestion of contaminated water or food
• Infective dose: 104 – 109
• Factors promoting transmission:
Hypochlorhydria, use of antacids, gastrectomy
Virulence factors
• Crossing of protective layer of mucus:
Active motility, mucinase ,proteolytic enzymes ,
hemagglutinin protease (cholera lectin)
• Adhesion: Toxin coregulated pilus (TCP)
• Cholera toxin
Clinical Manifestations
• Incubation period: 24 to 48hrs
• Sudden onset of painless watery diarrhea, quickly becomes
voluminous and followed shortly by vomiting
• Severe case: stool volume exceed 250ml/kg in first 24hrs
• Fluids and electrolytes not replaced hypovolemic shock and death
• Fever absent usually, muscle cramps common
• Stool: nonbilious, gray, slightly cloudy, with flecks of mucus, no blood
• “Rice- water stool”
• Ranges from asymptomatic to the most severe form, “cholera gravis”
75% asymptomatic
20% mild disease
2-5% severe

• Cholera gravis(Severe dehydration):

lethargic, unresponsive
 sunken eyes
 markedly decreased skin turgor
 cold, clammy skin
 dry mucus membrane

• Clinical features of V.cholerae O1 and O139 are indistinguishable

• Disease caused by Classical biotype V.cholerae O1 is more severe than
O1 El Tor biotype strains
Diagnosis
• Cholera should be considered in any area of the world,
when an individual aged 5yrs or older develops severe dehydration or
dies from acute watery diarrhea

• And, when an individual aged 2yrs or older develops acute watery

diarrhea in an area of the world known to be endemic for cholera

• Confirmed by identification of V.cholerae in stool specimen

Laboratory Diagnosis
Specimens
• Stool: freshly collected watery stool, collected
before starting antibiotics
• Rectal swab: Convalescent patients or carriers

Transport media
• Specimens transported as soon as possible
• If delay expected, 1-3ml of stool is mixed with 10-20ml of transport
media
• Venkatraman-Ramakrishnan (VR) medium
• Cary-Blair medium
Direct Microscopy
• Gram staining of mucus flakes of feces 
short, curved, gram negative bacilli , arranged
in parallel rows
• Fish in stream appearance, as described by
Koch
• Motility testing by hanging drop: Darting
motility, shooting star motility
• Dark field microscopy
Culture
• Peptone water: surface pellicle
• Grows optimally at 37°C
• Growth better in alkaline medium(pH 8.2)
Nutrient Agar
• Enrichment media:
Alkaline Peptone water
Monsur’s tellurite taurocholate peptone water
• Selective media:
Thiosulfate citrate bile salt sucrose (TCBS) agar
Monsur’s gelatin taurocholate trypticase
tellurite agar (GTTTA)
Blood Agar
 Biochemical Reactions
• Catalase and oxidase positive
• Triple sugar iron agar: A-/A-
• Sulfide Indole motility: S- I + M +
• Citrate: Utilized
• Urease: not produced
TCBS agar
• Nitrate reduction test is positive
• MR/VP: MR +/ VP+ ( - for classical biotype)
• Glucose, Sucrose, Mannitol fermented
• LAO: Only Lysine/ Ornithine decarboxylated
• String test : Positive
• Cholera red reaction: Positive

GTTT agar
Differences between Classical and El Tor
V.cholerae
Biotypes of V.cholerae O1 Classical biotype El Tor biotype

Β- hemolysis on sheep blood agar Negative Positive

Polymixin B (50IU) Susceptible Resistant

VP ( Voges Proskauer) test Negative Positive

CAMP test Negative Positive

Chick erythrocyte agglutination Negative Positive

Group IV phage susceptibility Susceptible Resistant

El Tor Phage V susceptibility Resistant Susceptible

Procedure for recovery of Vibrio cholerae from fecal specimens

Direct Enrichment

APW
6-8 hr, 35°C -37°C
TCBS

Optional screening
Nonselective
Tests
medium
String
Oxidase
V. cholerae O1 TSI
Polyvalent antisera Arginine or
Lysine

Ogawa and
Inaba antisera
Diagnostic Methods

1. Immunofluorescence
• Uses antisera conjugated to fluorescein isothiocyanate to visualize V.
cholerae O1
• Require expensive equipment, high quality immunologic reagents

2. Latex agglutination
• Detects the organism directly in fecal specimens
• The kit uses latex particles coated with monoclonal antibodies directed against
the A, B and C antigens of V. cholerae O1
• Sensitivity: 63%, Specificity: 88%
3. Coagglutination
• Detection of Vibrio cholerae O1 antigen from fecal sample
• Antibodies against V. cholerae O1 are bound to the surface of
Staphylococcus aureus (Cowan 1) cells
• In a positive reaction, staphylococcal cells are bound together in a
lattice-like arrangement
• Problems due to substances in stool which nonspecifically inhibit
agglutination and lattice formation of staphylococcal cells
• Cholera Screen- monoclonal antibody based coagglutination test
SMARTTM
• Rapid colorimetric immunoassay designed to detect the O1 antigen of V. cholerae in whole stool samples
• Monoclonal antibody-polyclonal antibody sandwich principle
• 95-100% sensitive, 97-100% specific

Institut Pasteur dipstick

• Detection of the V. cholerae O1 and O139 lipopolysaccharides (LPS) in stool samples
• Uses monoclonal antibodies specific to V.cholerae O1 and O139 LPS
• V.cholerae O1 sensitivity 94%, specificity 84 %
• V.cholerae O139 sensitivity 99%, specificity 96 %
• Through a licensure agreement, the RDT is now being produced by Span Diagnostics (India) under the trade name
Crystal VC™

SD Bioline Cholera AgO1/O139

• Vibrio cholerae O1 sensitivity 95%, specificity 94 %
• Vibrio cholerae O139 sensitivity 99%, specificity 98 %
 WHO guidelines on use of RDT for detection and
surveillance of cholera (2016)
• RDTs are intended to be used at primary health care level for surveillance purposes
• Used for:
early outbreak detection, as a tool for an initial alert,
monitoring of outbreaks,
monitoring of seasonal peaks in highly endemic areas

• In areas where confirmed cholera cases have not been recently reported, if one or
more patient(s) clinically suspect of cholera return a positive RDT result, this is
sufficient to immediately launch a cholera alert, send stool specimen to the
reference laboratory for confirmation, and initiate response measures (e.g. inform
authorities, mobilize resources and material, etc.)
• In areas with ongoing outbreaks, positive RDTs can be used to select
stool specimens from suspected cases for culture.

• RDTs are not a substitute for stool culture: any positive RDT(s) result
must be confirmed by culture or PCR as soon as possible before
confirming the alert and declaring a cholera outbreak.

• If all RDTs are negative, cholera should be ruled out.

• Cholera RDTs are of limited usefulness for individual diagnosis among

suspected cholera patients, since the results of the test would have
no influence on the immediate management of the case.
Treatment
• Rapid and adequate replacement of fluids and electrolytes
• Mortality rate for appropriately treated disease is usually <1%
• Fluid replacement orally using oral rehydration salts(ORS)
• IV fluids for severely dehydrated patients
• Ringer’s Lactate is the best choice

• Antibiotics are adjunct to hydration treatment

reduces volume of stool output
shortens duration of diarrhea
decreases bacterial shedding in feces
Use only in moderately and severely ill patients
 How to Approach Rehydration in Patients with Suspected Cholera
DEGREE OF DEHYDRATION
Some Moderate Severe
Assess degree Mentation Alert Restless, irritable Lethargic or unconscious
of Eyes Normal Sunken Sunken
Dehydration Skin turgor Normal recoil Slow recoil Very slow recoil (>2 sec)
Pulse Normal Rapid, low volume Weak or absent
Thirst Drinks normally Thirsty, drinks eagerly Drinks poorly or unable to drink

Approach to Fluid replacement Ongoing losses only 75 mL/kg in addition to >100 mL/kg in addition to ongoing
rehydration ongoing losses losses

Preferred route of Oral’ Oral or intravenous Intravenous

administration

Timing Usually guided by thirst Replace fluids over 3-4 hr As rapidly as possible until
circulation is restored;
Complete the remainder of
Fluids within 3 hr

Monitoring Observe until assured ongoing Observe every, 1-2 hr until all Once circulation is established,
losses can be adequately signs of dehydration resolve monitor every 1-2 hr
replaced by ORS and patient urinates
 Composition of Cholera Stools and Therapeutic Fluids for Cholera

CONCENTRRATION (mmoles/L)
Na+ K+ Cl- HCO3- Carbohydrate
Intravenous LRS 130 4 109 28 -(278 if DSLR
fluid available)
Normal Saline 154 0 154 0 -
Cholera Saline (“Dhaka solution”) 133 13 154 48 140
Oral ORS (WHO 2002) 75 20 65 10 (citrate) 75 (glucose)
rehydration Rice-based ORS 75 20 65 10 (citrate) 27 G rice syrup solids
therapy Homemade ORS ≈75 0 ≈75 0 ≈75
Electrolyte Cholera stool, adult 130 20 100 45 -
losses in Cholera stool, child 100 30 90 30 -
stools Noncholera stool, child (ETEC) 50 35 25 20 -
(composite
estimates)
 Antimicrobial Options for Treating Patients with cholera
CLASS ANTIMICROBIAL PEDIATRIC DOSE ADULT DOSE

Macrolide Erythromycin 12.5mg/kg/dose QID×3days 250mg QID×3days

Azithromycin 20mg/kg single dose 1gm single dose

Fluoroquinolones Ciprofloxacin 15mg/kg/dose BID×3days 500mg BID×3days

Tetracyclines Tetracycline 12.5mg/kg/dose QID ×3days 500mg QID × 3days

Doxycycline 4-6mg/kg × single dose 300mg × single dose

Prevention
• Provision of safe water
• Facilities for sanitary disposal of feces
• Improved nutrition
• Attention to food preparation and storage
3 Oral Cholera Vaccines are currently pre-qualified by WHO – Dukoral®, Shanchol™ and Euvichol®
Dukoral Shanchol and Euvichol
monovalent inactivated vaccine bivalent inactivated vaccine containing
containing killed whole cells of V. killed whole cells of V. cholerae O1
cholerae O1 plus additional recombinant and V. choleraeO139
cholera toxin B subunit
No. of doses 2 (3doses for 2-5yrs) 2
Volume 3ml 1.5 ml
Buffer solution for administration Required Not required
Protection against V. cholerae O1 V. cholerae O1 and O139
Age group > 2yrs >1 yr
Gap between doses minimum 1 wks minimum 2 wks
Protection of >50% for at least 2 yrs, Both vaccines provide sustained
induces an immune response relatively protection of >60% for at least 3 yrs
quickly after 2 doses
(7-10 days after the 2nd dose) A single dose has been proven to give
and has a good safety profile good short term protection for at least
6 months
and has a good safety profile