Seminar

Cholera
John D Clemens, G Balakrish Nair, Tahmeed Ahmed, Firdausi Qadri, Jan Holmgren

Cholera is an acute, watery diarrhoeal disease caused by Vibrio cholerae of the O1 or O139 serogroups. In the past Lancet 2017; 390: 1539–49
two centuries, cholera has emerged and spread from the Ganges Delta six times and from Indonesia once to cause Published Online
global pandemics. Rational approaches to the case management of cholera with oral and intravenous rehydration March 13, 2017
http://dx.doi.org/10.1016/
therapy have reduced the case fatality of cholera from more than 50% to much less than 1%. Despite improvements
S0140-6736(17)30559-7
in water quality, sanitation, and hygiene, as well as in the clinical treatment of cholera, the disease is still estimated to
International Centre for
cause about 100 000 deaths every year. Most deaths occur in cholera-endemic settings, and virtually all deaths occur in Diarrhoeal Disease Research,
developing countries. Contemporary understanding of immune protection against cholera, which results from local Bangladesh, Centre for Health
intestinal immunity, has yielded safe and protective orally administered cholera vaccines that are now globally and Population Research,
Dhaka, Bangladesh
stockpiled for use in the control of both epidemic and endemic cholera.
(Prof J D Clemens MD,
Prof T Ahmed MD,
Introduction Bacteriology Prof F Qadri PhD); UCLA Fielding
Cholera, an acute watery diarrheal disease caused by the V cholerae belongs to the Vibrionaceae family, whose School of Public Health,
Los Angeles, CA, USA
bacterium Vibrio cholerae, dates back to antiquity.1,2 In ecological niche is in saline coastal waters and estuaries,
(Prof J D Clemens); Korea
lieu of bacteriological confirmation, there is disagreement often in association with zooplankton and shellfish.16–19 University School of Medicine,
as to whether or not cholera in antiquity was exclusively These organisms can enter a viable but non-culturable Seoul, Korea (Prof J D Clemens);
an Asiatic disease, occurring in the Ganges Delta. What state within biofilms in response to nutrient deficiencies, WHO, Delhi, India
(Prof G B Nair PhD);
is clear, however, is that cholera has spread beyond Asia but they can proliferate during zooplankton blooms in and University of Goteborg,
during the course of seven recorded pandemics; the first response to the proper environmental conditions.16 Goteborg, Sweden
beginning in 1817, with ensuing pandemics in 1829, Chitin is the predominant source of carbon and nitrogen (Prof J Holmgren MD)
1852, 1863, 1881, 1889, and 1961—the most recent for cholera vibrios; chitin also induces natural Correspondence to:
persisting until the present. Each pandemic had a unique competence facilitating lateral gene transfer.20 Prof John D Clemens,
International Centre for
geographical spread, but together they ultimately More than 200 serogroups of V cholerae exist.
Diarrhoeal Disease Research,
included most of Asia, Africa, Europe, Australia, and the Serogroup O1 is further classified as sub-serotypes Inaba Bangladesh, Centre for Health
Americas.1,3,4 and Ogawa (and an unstable serotype termed Hikojima) and Population Research,
During the third pandemic, which reached Britain in on the basis of O-methylation of the O antigen. The GPO Box 128, Dhaka, Bangladesh
jclemens@icddrb.org
1853, the physician John Snow made his seminal O1 serogroup is also classified into the classical and
observations of the waterborne transmission of cholera.5 El Tor biotypes on the basis of phenotypic and genetic
The German microbiologist, Robert Koch, is often markers. Only vibrios that secrete cholera toxin can cause
credited with having first identified V cholerae (which he epidemics. The genes for cholera toxin are encoded by a
referred to as the Kommabacillus) as the causative filamentous bacteriophage, which differs slightly
organism for human disease during the fifth pandemic between classical and El Tor biotypes.21
in Egypt in 1883.6 However, Koch’s discovery appears to Every cholera pandemic is documented or presumed to
have been preceded by that of Italian microbiologist have been caused by serogroup O1 organisms, but only
Filippo Pacini.4 the first six pandemics were caused by the classical
The current, seventh pandemic has been caused by the biotype.22 In 1992, the emergence of a new serogroup,
El Tor biotype, first isolated in 1905 at the El Tor O139, caused a major outbreak that spanned most of Asia.
quarantine station in Egypt.7 In 1961, cholera caused by The O139 serogroup was shown to be genetically derived
this organism was reported in Java and Samarang, from El Tor biotype strains from the seventh pandemic,
Indonesia, from where it spread to much of Asia in the with genetic modification of the lipopolysaccharide and
1960s; to Africa, parts of the former USSR, the Middle the addition of a capsule to the parent strains surmised to
East, and southern Europe in the 1970s; to Latin America have occurred via horizontal gene transfer.23,24 Serogroup
in the early 1990s; and to Hispaniola in 2010.7–14 O1 El Tor organisms, of both Inaba and Ogawa serotypes,
Today, the global burden of cholera is high, and Africa
seems to be the major locus for this disease burden. An
analysis suggests that approximately 2·9 million cases Search strategy and selection criteria
and 95 000 deaths occur annually in countries with We searched PubMed for papers published from Jan 1, 2010,
endemic cholera, with 60% of cases and 68% of deaths to Feb 1, 2016, using the terms “cholera” and “Vibrio
recorded in Africa.15 cholerae”. We also referred to work in our personal collections
Since the previous Lancet Seminar in 2012,3 important of original research papers and reviews from the period
advances have been made, especially in our understanding 1966–2016. We gave similar emphasis to important
of human genetic susceptibility to cholera and the role of publications during the past 5 years and to pivotal older
the microbiome in cholera pathogenesis, and in the publications.
global deployment of the first low-cost cholera vaccine.

www.thelancet.com Vol 390 September 23, 2017 1539

Downloaded for Anonymous User (n/a) at Riga Stradins University from ClinicalKey.com by Elsevier on October 12, 2018.
For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved.
 Seminar
now account for virtually all cholera cases worldwide, and
the O139 strain is rarely isolated.
El Tor strains have been classified into four major
clonal groups: the Australian clone; the US Gulf Coast
clone; the seventh pandemic clone; and the Latin
American clone.25 Epidemiological mapping is being
increasingly replaced by robust high-resolution markers
through identification of single nucleotide
polymorphisms in whole genomes. These developments
have led to the identification of variants of El Tor biotype
that share phenotypic characteristics of both El Tor and
classical biotypes. These variant El Tor biotype strains
have phenotypic characteristics that classify them as
El Tor, but they secrete classical-type enterotoxin. Since
2002, these variant strains have displaced typical El Tor
biotype in Bangladesh and several other sites in Asia and
Africa.26 The strains causing the 2010 outbreak of cholera
in Haiti, which originated in Asia, also show features of
the variant strains.27 Certain variant El Tor biotype strains
seem to cause more severe disease than typical El Tor
biotype strains, probably because of an increased
secretion of cholera toxin.28
Epidemiology
Only a small proportion of cholera infections are
symptomatic, the proportion being affected by factors
such as the ingested inoculum, V cholerae biotype, and
pre-existing host anticholera immunity.1 Cholera is
classified as either endemic or epidemic. Endemic
cholera refers to the repeated occurrence of cholera in a
population during a period of time, without the need for
external reintroduction. WHO defines a cholera-endemic
population as one in which cholera has been confirmed
from faecal sample culture in at least 3 of the past
5 years.29 In populations with endemic cholera, outbreaks
typically occur seasonally, and children younger than
5 years of age are at highest risk because of their low
levels of pre-existing natural anti-cholera immunity.30
Epidemic cholera, by contrast, refers to cholera that
requires exogenous introduction of cholera vibrios into a
population. In such situations, incidence rates of cholera
are usually age independent, and, since affected
populations often lack pre-existing immunity to cholera,
the clinical range of cholera illnesses can be more
severe.23 Although useful conceptually, the terms
endemic and epidemic cholera represent two ends of a
spectrum, and large outbreaks termed as epidemics can
occur in populations with endemic cholera.
V cholerae spreads through the faecal–oral route, and a
high infectious dose (10⁸ organisms) is necessary to
cause illness in experimentally challenged, healthy
volunteers, although lower doses might cause disease in
individuals whose gastric acid barrier is impaired or
when the bacteria are coingested with foods that buffer
gastric acid.31 Person-to-person transmission is well
documented within households.32 V cholerae organisms
are present in human stool as both individual cells and as
1540 www.thelancet.com Vol 390 September 23, 2017
Downloaded for Anonymous User (n/a) at Riga Stradins University from ClinicalKey.com by Elsevier on October 12, 2018.
For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved.
biofilm-like aggregates.33,34 Infected persons generally
excrete the bacteria for only a few days, although people
with symptoms might shed bacteria for up to 2 weeks or,
in rare cases, even longer.35 Patients passing rice water
stool excrete hyperinfective vibrios, which might increase
transmission of disease.36
Under proper conditions of surface water temperature
and terrestrial nutrient discharge, zooplankton and
phytoplankton associated with cholera vibrios may
proliferate and thereby increase the number of active
cholera bacteria.16 Epidemiological patterns of cholera in
human populations relate to their geographical proximity
to such waters, short-cycle and long-cycle climatic cycles,
natural disasters such as floods, cyclones, and
earthquakes, and both immune and non-immune host
factors in affected populations.37–39 Lytic bacteriophages,
both in the environment and in the stools of patients
with cholera, might limit the course of individual
patients’ illnesses and outbreaks at the population level.40
Results of a genomic analysis of strains of the seventh
pandemic revealed that strains arose from a single source
in the Bay of Bengal, presumably after initial importation
from Indonesia, and then spread to distant sites in at
least three independent but overlapping waves.41 The
second and third waves were due to organisms that
carried a conjugative, self-transmissible, chromosomally
integrating SXT element that conferred resistance to
multiple antibiotics.42 Isolates from the massive outbreak
of cholera that began in Haiti in 2010 resemble those
from the third wave, supporting the hypothesis that this
outbreak resulted from the spread of V cholerae into
sources of drinking water by UN troops dispatched to
Haiti from Nepal.27
Virulence factors and pathogenesis
To adapt to such diverse milieus as the human intestine
and various marine environments, V cholerae O1 (and
O139) has refined systems to rapidly modulate their gene
expression in response to environmental changes upon
entering the human body. Bacterial colonisation and the
production and action of cholera toxin in the small
intestine are important pathogenic events in cholera
infection and disease (figure 1). Although these bacteria
are dividing rapidly throughout the whole intestine of
patients with cholera, results of intestinal perfusion
studies show that as much as 80–90 % of cholera fluid
secretion occurs in the first metre of the intestine, with
the electrolyte loss from the jejunum exceeding that
from the ileum.44,45
The ability of V cholerae O1 to produce an exotoxin that
induces fluid secretion in the infected small intestine
was first described in rabbits in 1959.46 Intense research
in the 1970s identified the subunit structure and function
of cholera toxin,47 its cell membrane receptor,48 and its
ability to induce intracellular cyclic AMP signalling via
ADP-ribosylation of adenylate cyclase and thereby trigger
fluid secretion by the intestinal epithelium.49–52 The
 Seminar

Vibrio cholerae

Mucus

CTA-1
NaCI
CTA-2 Cholera toxin
Epithelial cell
CTB
GM1

CI–

CFTR ATP
Lipid raft AC
PKA [cAMP]
NAD ADPR Gsα
Endoplasmic reticulum
CTB CTA CTA-1
Degradasome

Golgi

Figure 1: Cholera pathogenesis and cholera toxin action

After ingestion, Vibrio cholerae colonise the small intestine and secrete cholera toxin, which has a doughnut-like structure with a central enzymatic toxic-active A
(CTA-1 + CTA-2) subunit associated with a pentameric B subunit (CTB). After binding to GM1 ganglioside receptors on small intestinal epithelial cells, which are
mainly localised in lipid rafts on the cell surface, the cholera toxin is endocytosed and transported to the degradasome via the endoplasmic reticulum via a retrograde
pathway, which, dependent on cell type, may or may not involve passage through the Golgi apparatus. In the endoplasmic reticulum, CTA dissociates from CTB,
allowing CTA-1 to reach the cytosol by being translocated through the degradasome pathway. In the cytosol CTA-1 subunits rapidly refold and bind to the Gsα
subunit of adenylate cyclase in the cell membrane; upon binding, CTA-1 ADP-ribosylates the Gsα subunit, which stimulates adenylate cyclase activity, leading to an
increase in intracellular concentration of cyclic AMP, activation of protein kinase A, phosphorylation of the cystic fibrosis transmembrane conductance regulator, a
major chloride channel, and extracellular secretion of chloride ions and water. Cholera toxin-induced chloride (and bicarbonate) ion secretion is particularly
pronounced in intestinal crypt cells, whereas the increased intracellular cyclic AMP concentrations in villus cells mainly inhibit the uptake of NaCl and water.
ADPR=ADP ribose. AC=adenylate cyclase. cAMP=cyclic AMP. CFTR=cystic fibrosis transmembrane conductance regulator. Cl–=chloride ion. NaCl=sodium chloride.
Gsα=G subunit of adenylate cylcase. PKA=protein kinase A. Adapted from Clemens and colleagues,43 by permission of Nature Publishing Group.

84 kDa cholera toxin consists of a single 28 kDa toxic-
active A subunit non-covalently attached via its carboxy-
end tail (CTA-2) to a ring of five identical 11·6 kDa
cell-binding B subunits. After assembly in the periplasm,
cholera toxin is efficiently secreted from V cholerae.53 In
the intestine, cholera toxin binds via its B subunits with
high affinity to the epithelial cell surface receptor,
monosialoganglioside GM1 [Gal(β1-3)GalNac(β1-4)
(NeuAc(α2-3)Gal(β1-4)Glc]→ceramide], which was the
first human cell surface receptor to be chemically
identified and structurally defined. After binding to GM1
(localised mainly in lipid rafts on the cell surface), cholera
toxin is endocytosed and transported to the endoplasmic
reticulum via a retrograde transport pathway.54
In the endoplasmic reticulum, the major part of the
A subunit (CTA-1) dissociates from the B pentamer to
enter the cytosol,55 where CTA-1 catalyses ADP–
ribosylation of the Gsα subunit of adenylate cyclase
inside the cell membrane. This reaction locks adenylate
cyclase in a GTP-bound activated state, resulting in
enhanced cyclic AMP (cAMP) production. The increase
in intracellular cAMP concentration causes an imbalance
in electrolyte transport across the epithelial cell
membrane, with a decrease in sodium uptake and an
increase in chloride and bicarbonate export, largely by
stimulating the cystic fibrosis transmembrane
conductance regulator (CFTR) chloride channel via
cAMP-responsive protein kinase.56 Water follows this ion
gradient to produce a net fluid loss. In addition to the
direct effect of cholera toxin on cAMP production in
enterocytes, toxin-induced secretion might have a
neurological component by inducing the release of the
secretagogue vasointestinal peptide from intestinal
neural networks.57
The mechanisms by which V cholerae colonises the
small intestine have remained more elusive than that of
the toxin action. Important features of V cholerae that
promote colonisation include: (1) a cholera toxin
coregulated pilus, which has been found to be important
in at least the early stage of colonisation;58,59 (2) HapA, a
soluble hemagglutinin/Zn-metalloprotease that facilitates
bacterial penetration of the intestinal mucus layer;60 and
(3) the flagellum that moves the bacteria towards the
epithelial surface.61
In addition to HapA, mucinase, and neuraminidase,
several toxins produced by V cholerae might contribute to

www.thelancet.com Vol 390 September 23, 2017 1541

Downloaded for Anonymous User (n/a) at Riga Stradins University from ClinicalKey.com by Elsevier on October 12, 2018.
For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved.
 Seminar
diarrhoea: Zot and Ace have enterotoxic activity and are
part of the CTXφ phage that encodes cholera toxin; RTX
toxin, an actin-crosslinking toxin produced by the El Tor
biotype but not classical strains; S-CEP, a cytotonic
protein that elongates Chinese hamster ovary cells; and
hemolysin, a pore-forming and vacuolating toxin.62
Finally, bacterial detachment and dissemination in the
later stages of the infectious cycle are associated with and
dependent on the repression of several of the same
factors. Beside the various host-related signals discussed
above, bacterial cell density is an important regulator of
virulence factor expression. In a process known as
quorum sensing, bacteria can monitor their population
density via expression of small autoinducer molecules. To
a great extent, the expression of cholera toxin coregulated
pilus, cholera toxin protein, and several other virulence
factors is under quorum sensing and HapR signalling
control. Thus, their expression is increased at low cell
density and repressed at high cell density.63
Host susceptibility and immunity
Susceptibility to infection by V cholerae depends both on
adaptive immune responses, induced by previous
infection or vaccination, and on innate host factors.
Stomach acidity and ABO blood groups are the innate
host factors that are most studied. Low gastric acid level
has been associated with more severe cholera disease.64
Case-control studies have found that blood group O
individuals are at increased risk of diarrhoea due to both
V cholerae O1 and V cholerae O139.65 Cholera is proposed
to have been a selection factor leading to a uniquely low
prevalence of blood group O in Bangladesh and west
Bengal.66 The correlation between blood group O and
disease severity in Bangladesh was subsequently found
to be restricted to cholera caused by the El Tor biotype.67
Findings from gene expression studies of duodenal
biopsy samples from patients with acute cholera revealed
that most early upregulated genes encode innate
immunity proteins, including several with antibacterial
activity, and that one particular group of genes seems to
be activated indirectly via cholera toxin-induced IL-1.68
The strongest upregulated gene encoded for LPLUNC1,
an antimicrobial protein, and polymorphism in the
LPLUNC1 gene was found to be associated with an
increased risk of cholera.69 Results of a study in
Bangladesh showed that genes related to NF-kB innate
immunity signalling and to potassium channel genes
involved in cyclic AMP-mediated chloride secretion were
associated with cholera risk and appeared to be under
natural selection in this population.70
Infants and young toddlers who are not breastfed at the
time of exposure to V cholerae seem to be at increased
risk of cholera, and retinol deficiency might be associated
with a higher risk in household contacts of cholera index
cases.71,72 The gut microbiome composition has been
associated with the recovery from cholera in Bangladeshi
adults.73 Findings from a study in Mozambique
1542 www.thelancet.com Vol 390 September 23, 2017
Downloaded for Anonymous User (n/a) at Riga Stradins University from ClinicalKey.com by Elsevier on October 12, 2018.
For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved.
implicated HIV co-infection as a risk factor for cholera,
raising the possibility of the convergence of the ongoing
pandemics of these two infections.74
Studies in experimentally infected volunteers and
epidemiologic data from the field suggest that an initial
clinical cholera infection confers protection against
subsequent, serogroup-homologous cholera episodes for
at least 3 years.75 Immune protection appears to be more
pronounced after cholera caused by the classical biotype
than after El Tor biotype and more long-lasting after
Inaba infection than after Ogawa serotype infection.75
The immune response in individuals with cholera is
directed primarily against bacterial surface molecules and
against cholera toxin. The response includes intestinal–
mucosal secretory IgA (SIgA) and serum IgA, IgG,
vibriocidal antibodies, antibody-secreting cells, T cells,
and, of special importance for long-term protection,
memory B cells and T cells.76–81 The best studied measure
of adaptive immunity to V cholerae is the level of serum
vibriocidal antibodies. In cholera-endemic areas,
complement-dependent vibriocidal (mainly IgM) antibody
titres increase progressively with age and correlate with a
reduced risk of cholera.82 However, serum vibriocidal
antibodies are only a proxy for the intestinal mucosal
immune status. Studies indicate that protective immunity
against cholera depends on the stimulation of a mucosal
immune response in the intestine and is mediated by
antibacterial and antitoxin SIgA antibodies.83,84
Protective antibacterial immunity against experimental
cholera caused by O1 bacteria is mainly due to antibodies to
the O1 lipopolysaccharide.83,84 The O1 lipopolysaccharide
contains major group-specific A epitopes shared between
the Inaba and Ogawa serotypes and additional serotype-
specific B epitopes (Ogawa) or C epitopes (Inaba). Both
cross-reactive and serotype-specific antibodies contribute to
protection.83 Protective immunity against V cholerae O139
also seems to be mediated predominantly by antibodies to
O139 lipopolysaccharide.85 The protective relevance, if any,
of other anti-bacterial antibodies, including antibodies
against toxin-coregulated pili, remains to be defined.
Findings from studies, both in animals and in human
beings, have shown a protective role for antitoxic
immunity.86–88 An important observation guiding the design
of oral cholera vaccines, especially the inactivated whole-
cell plus cholera toxin B subunit oral cholera vaccine, is the
combined roles of antibacterial and antitoxic immunity in
mediating protection. Antibodies against lipopolysaccharide
and cholera toxin B subunit can independently protect
against disease by inhibiting bacterial colonisation and
toxin binding, respectively, and when present together in
the gut, their protective effects are strongly synergistic.88
Diagnosis
Microbiological culture of stool samples or rectal swabs
for the isolation and identification of V cholerae is
considered the gold standard for confirmation of cholera
diagnosis. Faecal specimens are plated directly on
 Seminar

selective media, containing either thiosulphate citrate absence of rehydration therapy. Depending on the
bile salts, sucrose, or taurocholate-tellurite gelatin agar, severity of dehydration, a patient can have thirst and
as well as after enrichment in alkaline peptone water. irritability and, in later stages, lethargy, a rapid radial
Depending on the medium used, colonies are tested pulse, loss of skin turgor, diminished urine output, low
directly or after culture on non-selective media by slide blood pressure, rapid breathing, and sunken eyes.102
agglutination with different monoclonal or polyclonal When severely dehydrated, a patient can progress to
antibodies to detect V cholerae O1 and O139.89,90 hypovolemic shock.
Isolation of V cholerae by microbiological culture Complications of cholera include electrolyte
requires competent laboratory support and can take up to disturbances, including hypokalaemia, hyponatraemia,
2 days after collection of faecal specimens. Dark field and hypocalcaemia (sometimes associated with muscle
microscopy is a useful technique for rapid detection of cramps and relieved by intravenous administration of
V cholerae in stool samples because of the characteristic calcium gluconate), and acidosis. Children can develop
motility of cholera vibrios and inhibition of the hypoglycaemia due to depleted hepatic glycogen
movement using antibodies that are specific for reserves and insufficient gluconeogenesis, which may
V cholerae. This test has a sensitivity of approximately cause seizures. Other complications include various
50% by comparison with culture.34,91 Other rapid manifestations of diminished perfusion of end organs,
diagnostic tests have been developed with the intention including acute renal failure, stroke, and, in pregnant
of providing immediate, point-of-care diagnoses, ideally patients, miscarriages, premature deliveries, and
at low cost and with implementation by semi-skilled stillbirths.4,103,104
professionals. At least 24 rapid diagnostic tests have been The immediate threat to the survival of a patient with
developed to date, employing immunological (eg, ELISA, severe cholera is hypovolemic shock. Consequently, the
coagglutination, lateral flow system) or molecular (eg, initial clinical assessment of a patient with cholera should
multiplex or reverse transcriptase PCR, nucleic acid focus on determining the level of dehydration and
lateral flow biosensor) platforms. To date, only a few estimating the volume of body fluid that needs replacement,
tests, including the coagglutination test, the Sensitive in addition to ongoing fluid losses. This assessment is
Membrane Antigen Rapid Test, the Institut Pasteur and based on clinical signs and symptoms (table 1).105,106
Crystal VC rapid dipsticks, and Medicos, have been Additional replacement of ongoing fluid losses in patients
found to be potentially suitable for public health with severe purging is greatly assisted by use of a cholera
application, with the performance of the rapid dipstick cot, a simple cot with a central hole and plastic sleeve
augmented when specimens are enriched in alkaline.92–96 draining into a collection bucket that allows ongoing
Multiplex PCR assay targeting the cholera toxin gene measurement of stool output (figure 2). In the absence of
(ctxA) and the O1 and O139-specific rfb gene is used in such cots, ongoing losses can be estimated as 10–20 mL/kg
certain laboratory settings.97 The TaqMan Array Card for each diarrhoeal stool or episode of vomiting.3
system, using a quantitative RT-PCR format, has been
found to have a sensitivity and specificity of 85% and 77%, No dehydration Some dehydration Severe dehydration
respectively, for detection of V cholerae in comparison with Clinical assessment criteria
conventional methods, and 98% and 96%, respectively, General condition Normal Irritable; less active Lethargic or comatose
when compared with other PCR-Luminex assays.97–99
Eyes* Normal Sunken ··
Mucosa Normal Dry ··
Clinical features and management Thirst Normal Thirsty Unable to drink
In its most severe form, cholera is known for its acute
Skin turgor Normal (quick return Reduced (slow return to ··
nature that leads to severe dehydration within hours and to original position) normal position)
death if not treated adequately.100 Before the establishment Radial pulse Normal Reduced or rapid Uncountable or absent
of modern approaches to rehydration therapy, case fatality Synthesis of criteria Does not meet Has at least two signs of Meets criteria for some
rates in excess of 50% were reported.1,101 Application of criteria for either some dehydration, one of dehydration plus at least
modern approaches to clinical management can lower some or severe which is irritability, thirst, or one of the clinical criteria
dehydration reduced skin turgor for severe dehydration
the case fatality rate to well less than 1%.
Fluid deficit (percentage <5% 5–10% >10%
The clinical features of cholera can range from mild to
of bodyweight)
very severe. After an incubation period of approximately
Rehydration therapy Replace ongoing 50–100 mL/kg and replace >100 mL/kg and replace
18 h to 5 days, the illness typically starts suddenly with fluid losses only (oral ongoing losses (intravenous ongoing losses
passage of watery stools and vomiting.1 With continued fluids ad libitum) fluids, oral fluids, or both (intravenous fluids
purging, the stools can assume the appearance of rice initially, then oral fluids) initially, then oral fluids)
water with flakes of mucus, often with a fishy odour. *In some infants and children the eyes normally appear somewhat sunken, so it is helpful to ask the mother if the
Systemic manifestations such as fever are absent unless child’s eyes are normal or more sunken than usual.
there is a co-infection.
Table 1: Criteria for estimating the extent of dehydration and formulating initial management of
The most salient features of the illness are those dehydration in patients with cholera, using the Dhaka Method105–107
related to dehydration, which can rapidly ensue in the

www.thelancet.com Vol 390 September 23, 2017 1543

Downloaded for Anonymous User (n/a) at Riga Stradins University from ClinicalKey.com by Elsevier on October 12, 2018.
For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved.
 Seminar
Figure 2: Cholera cot beds and intravenous fluids in the emergency resuscitation unit of the hospital of the
International Centre for Diarrhoeal Disease Research, Bangladesh, Dhaka, Bangladesh
Severe dehydration is a medical emergency, and
intravenous rehydration should be administered
aggressively to restore circulation. In a busy hospital,
shock is diagnosed by looking at the capillary refill
(pressing the nail bed of the thumb and leaving it for the
colour to return; a delayed return of blood by more than
3 s reflects prolonged capillary refill and is a sign of
shock)105 and assessing the radial pulse volume and blood
pressure. In patients with severe dehydration on
presentation, infants should receive 30 mL/kg
intravenously in the first hour in addition to a volume
that replaces ongoing losses, followed by 70 mL/kg during
the next 5 h, whereas older patients should receive
30 mL/kg in the first 30 min in addition to a volume that
replaces ongoing losses, followed by 70 mL/kg over the
next 2·5 hours. Patients with some dehydration should
receive 50–100 mL/kg in addition to volumes that replace
ongoing losses within the initial 3–4 hours.
The intravenous solution to be used is either Ringer’s
lactate (Na+ 130 mmol/L, K+ 4 mmol/L, Ca2+ 3 mmol/L,
Cl– 109 mmol/L, lactate 28 mmol/L) or Cholera Saline
(Na+ 133 mmol/L, K+ 13 mmol/L, Cl- 98 mmol/L,
acetate 48 mmol/L). The first part of the infusion
(30 mL/kg) for severe dehydration can be repeated if the
radial pulse remains weak or undetectable. For patients
with some dehydration, initial management might
deploy intravenous fluids, oral rehydration solution, or
both, depending on the ability of the patient to ingest the
oral rehydration solution and the magnitude of ongoing
fluid losses. In any case, oral fluids given as oral
rehydration solution should be started as soon as the
patient is capable of drinking.
Unless ongoing losses are very large (ie,
10 mL/kg per hour or more) or the patient is unable to
ingest fluids, patients presenting without evidence of
1544 www.thelancet.com Vol 390 September 23, 2017
Downloaded for Anonymous User (n/a) at Riga Stradins University from ClinicalKey.com by Elsevier on October 12, 2018.
For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved.
dehydration can be managed with oral rehydration
solution alone. The oral rehydration solution that is
recommended by WHO contains glucose as a source of
carbohydrates and has reduced osmolarity, which is
associated with reduced stool output. Compared with
glucose-based oral rehydration solutions, rice-based
solutions have been shown to be more effective in
reducing the volume of stool losses and the duration of
diarrhoea in cases of severe cholera.108 If no such solution
is available, an oral rehydration solution can be prepared
by mixing half a teaspoon of salt with six teaspoons of
sugar in 1 L of water.
Rehydration of children requires consideration of their
nutritional status. Children with severe acute
malnutrition (weight-for-length less than –3 Z score,
bilateral pedal oedema, or mid-upper arm circumference
less than 115 mm) should be managed carefully to
prevent acute fluid overload and heart failure. The
rehydration process should take 10–12 hours.107,109–111
ReSoMal, the special oral rehydration solution for
children with severe acute malnutrition, has been found
to be associated with symptomatic hyponatraemia in
children who also have cholera. Instead, the standard
oral rehydration solution recommended by WHO should
be used.112
Vomiting is common in patients who are dehydrated
and acidotic, but usually subsides with appropriate
rehydration. Antiemetics have no role, and might
interfere with rehydration because of their sedating
effects. Antibiotic treatment, initiated after resolution of
the initial fluid deficit and cessation of vomiting, reduces
the total volume of stool passed and shortens the period
of faecal excretion of V cholerae.100,113 Antibiotic treatment
should be dictated by local antimicrobial susceptibility
profiles. Azithromycin and ciprofloxacin are commonly
used, although azithromycin has been shown to be more
effective than ciprofloxacin in terms of shortened
duration of diarrhoea, reduced stool volume, lower
frequency of vomiting, and cessation of faecal excretion
of cholera vibrios. When local isolates are sensitive to
azithromycin, children are given 20 mg/kg and adults
are given 1 g, each as a single dose regimen.114,115
Ciprofloxacin is given to children in a dose of 15 mg/kg
twice daily for 3 days, and to adults as a single 1 g dose.
In areas with low levels of tetracycline resistance, a single
dose of 300 mg doxycycline can be used for adults.
Prophylactic administration of antibiotics during
outbreaks or for travellers is not advised.
Except for breastfeeding, which should continue
alongside administration of oral rehydration solution,
food should not be given during the initial 4-hour
rehydration period to reduce the chance of vomiting and
to focus on rehydration, although feeding should be
resumed as soon as possible thereafter. Children with
severe acute malnutrition, however, should be fed within
this period to prevent hypoglycaemia. Zinc supple­
mentation reduces the duration and severity of the
 Seminar

episode of diarrhoea in children.116 Supplemental zinc used to prevent cholera in cholera-endemic populations
(10–20 mg) can be started as soon as the vomiting (table 2). Dukoral has been licensed for travellers. Each of
subsides and is given once daily for 10–14 days.117 these vaccines is licensed to be given as a two-dose or
three-dose regimen. One vaccine (Shanchol) has been
Prevention shown to protect for at least 5 years when given in a two-
Major improvements of municipal water systems, dose regimen and for at least 6 months when given as a
sanitation infrastructure, and hygienic conditions in single dose, although the latter was observed only in older
most industrialised countries during the late 19th and children and adults.136,137 Another inactivated whole-vibrio
early 20th centuries led to the elimination of indigenous vaccine (ORCVAX, VaBiotech, Vietnam) was introduced
transmission of cholera in these settings.118 In many in Vietnam in the 1990s, with several million doses
developing countries, implementation of such systems, administered, but this vaccine is licensed only in Vietnam.
with their attendant high costs, has not proved feasible Despite the availability of WHO-prequalified oral
until now and will probaby not be feasible in the near cholera vaccines, use of these vaccines has, until recently,
future. Although a UN report119 in 2015 reported that 91% been limited. In 1999, WHO recommended the pre-
of the world’s population had access to improved emptive use of oral cholera vaccine in emergency
drinking water sources, only 58% had access to piped situations in which a cholera outbreak was a high risk
water on premises, and only 68% of the population had and that a 2-million dose oral cholera vaccine stockpile be
access to an improved sanitation facility. Many simple established for use in endemic and emergency settings.138
and cheap approaches to improving drinking water at the For various reasons, including concerns about vaccine
source and at point of use, including water filtration and costs and logistical challenges of administering a two-
disinfection, as well as interventions to improve personal dose vaccine regimen, as well as optimism about the
hygiene, have been studied, but few studies have
documented an effect on the risk of cholera, and none of Recombinant cholera toxin B Whole bacterial cells only
these approaches has been demonstrated to be effective subunit–whole bacterial cells (Shanchol, Shantha Biotech;
at scale or sustainable in the long term.120–123 (Dukoral, Valneva) Euvichol, Eubiologics)
Parenteral vaccines against cholera have been in Cellular constituent O1 serogroup El Tor and classical O1 serogroup El Tor and classical
development since shortly after the discovery of the biotypes biotypes; O139 serogroup
etiologic cholera vibrio in the late 19th century. Although Number of doses in primary Two doses given 1–6 weeks apart Two doses given 14 days apart
regimen (three doses for children 2–5 years
early studies seemed to show that these vaccines were of age)
effective, appropriately controlled clinical trials in the Need for and frequency of After 2 years (every 6 months for Not specified
1960s and 1970s found that the vaccines conferred only booster vaccine children 2–5 years of age)
modest, short-lasting protection, often with side- Minimal age according to licence 2 years 1 year
effects.1,124 Together with compelling data on the Safety and tolerability High (also true for people with High (presumably also true for
effectiveness of oral and intravenous rehydration therapy HIV135) people with HIV, given the
for cholera, these findings led to the abandonment of similarity of the vaccine to Dukoral)
WHO recommendations for the public health use of Contraindicated for women No No
during pregnancy
these vaccines in the 1970s.
Time of onset of protection after No data available (presumably No data available (presumably
Scientific findings suggesting intestinal mucosal complete dosing 1 week) 1 week)
immunity as the basis for immune protection against Protective efficacy (%) 57% at 2 years after vaccination 65% at 5 years after vaccination
cholera and oral immunisation as the most efficient Protection greater in children Yes Yes
approach to eliciting this immunity led to the development older than 5 years than in
of modern oral vaccines against cholera.1,75,80,125 To date, younger children
both live, genetically attenuated, and inactivated oral Protection by biotype Greatest against classical biotype Data is available only for the El Tor
cholera vaccines have been developed;43,126–132 although the than El Tor biotype; also protects biotype; also protects against
against newly emergent hybrid newly emergent hybrid El Tor
live attenuated vaccines have not been successful in El Tor cholera cholera
protecting against cholera in endemic settings.133 Herd protection Yes Yes
However, live attenuated vaccine, CVD 103HgR, has Cross protection against Yes No
proven useful for travellers. A recent version of diarrhoea associated with heat-
CVD 103 HgR, Vaxchora, was recently licensed in the labile toxin–enterotoxigenic
Escherichia coli
USA for use by travellers aged 18–64 years.134 Inactivated
Requirement for Yes No
oral cholera vaccines have been successful in licensure
coadministration with liquid
and international acceptance. Three such vaccines buffer
containing inactivated cholera vibrios, either with cholera Storage temperature; shelf life 2–8°C; 3 years 2–8°C; 2 years
toxin B subunit (Dukoral, Valneva, Sweden) or without Negotiated price per dose for the US$5·25 (negotiated price for US$1·85 for Shanchol; the official
this moiety (Shanchol, Shantha Biotechnics, India; and public sector WHO) price of Euvichol is not known
Euvichol, Eubiologics, Korea), have been prequalified by
Table 2: Features of the three licensed and WHO-prequalified oral cholera vaccines125
WHO for purchase and use by UN agencies and can be

www.thelancet.com Vol 390 September 23, 2017 1545

Downloaded for Anonymous User (n/a) at Riga Stradins University from ClinicalKey.com by Elsevier on October 12, 2018.
For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved.
 Seminar
1·0 Unvaccinated
Vaccinated
0·8
B The use of oral cholera vaccines and implementation
Probability of diarrhoea
0·6
0·4
0·2 D (figure 3).147 In a recent large-scale, cluster-randomised
0
105 106 107 108 109 1010 1011
Number of ingested vibrios
A—B: Improved water quality, hygiene, and sanitation
A—C: Vaccination
A—D: Improved water quality, hygiene, and sanitation plus vaccination
Figure 3: Hypothetical relation between the effect of oral cholera vaccines
and improvements in water quality, hygiene, and sanitation on the risk of
cholera, by the size of ingested cholera inoculum
Two hypothetical curves show the ingested inoculum of cholera organisms in
relation to the probability of diarrhoea after ingestion. The top curve corresponds
to an unvaccinated individual, and the bottom curve corresponds to a vaccinated
individual. The top curve roughly describes the lower risk of symptomatic cholera
after interventions in water quality, hygiene, and sanitation, which act to decrease
the frequency or dose of ingestion of cholera vibrios, or both (movement from
state A to state B). The bottom curve reflects the same for vaccinated individuals
(movement from state C to state D). The effect of oral cholera vaccines should be
to decrease the probability of becoming ill at any (or at least most) ingested doses
(movement from state A to state C). Because of these relations, the effects of
interventions in water quality, hygiene, and sanitation and oral cholera vaccines
should combine to produce a greater preventive effect than either intervention
alone (movement from state A to state D). Reproduced from Clemens and
colleagues,147 by permission of Springer Verlag.
effect of near-term improvements in water and sanitation,
these recommendations were not implemented. This
optimism has not been borne out, as reflected by data
showing the unrelenting global burden of cholera,139
including the recent establishment of endemic cholera in
Haiti, after nearly a century during which cholera was
not reported in this country.140 Encouragingly, inactivated
oral cholera vaccines have been shown to confer herd
protection to populations as well as direct protection to
vaccinated individuals, indicating that these vaccines are
more impactful and cost-effective than previously
thought.141,142
In 2011, the 64th World Health Assembly called for an
integrated, comprehensive strategy of cholera prevention
and control, including the use of oral cholera vaccine,
where appropriate. Subsequently, WHO and GAVI, the
Vaccine Alliance, created a global stockpile of Shanchol
for use in cholera outbreaks.143 To date, more than
4 million doses of Shanchol have been deployed from the
stockpile in both cholera-endemic and cholera-epidemic
settings, including ten countries in Africa and one
country each in Asia, the Middle East, and the Caribbean.
Vaccine delivery has consistently been found to be
logistically feasible, safe, and acceptable, and has been
observed to confer levels of protection similar to or
higher than those reported in a pivotal efficacy trial in
Kolkata, India.144–146 As supply of stockpiled vaccines
1546 www.thelancet.com Vol 390 September 23, 2017
Downloaded for Anonymous User (n/a) at Riga Stradins University from ClinicalKey.com by Elsevier on October 12, 2018.
For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved.
increases, the use of the stockpile is projected by GAVI to
A expand in the coming years.
of water, sanitation, and hygiene interventions have in
the past been viewed as in competition with one another.
There is now a better appreciation of a plausible
C
theoretical basis on which to consider these
interventions as complementary, if not synergistic
community effectiveness trial in Dhaka, Bangladesh,
Shanchol was tested alone versus in conjunction
1012 1013 with concomitant water, sanitation, and hygiene
interventions; results showed no additional prevention
against cholera by water, sanitation, and hygiene
interventions, although this might have reflected the
less than optimal uptake of the water, sanitation, and
hygiene interventions.146 Further exploration of the
preventive effect of combined oral cholera vaccine and
water, sanitation, and hygiene interventions is an
important priority for the future.
Contributors
JDC coordinated the overall preparation of this paper and wrote the initial
draft of the sections about epidemiology and prevention. GBN wrote the
initial draft of the section about bacteriology. TA wrote the initial draft of
the section about clinical features and management. FQ wrote the initial
draft of the section about diagnosis. JH wrote the initial draft of the section
about virulence factors and pathogenesis and about host susceptibility and
immunity. All authors reviewed, edited, and approved the final version of
the manuscript.
Declaration of interests
We declare no competing interests. Jocalyn Clark assisted with the
manuscript in her previous role at the International Centre for
Diarrhoeal Disease Research, Bangladesh, before she became an
Executive Editor at The Lancet.
Acknowledgments
This work was supported by core grants to the International Centre for
Diarrhoeal Disease Research, Bangladesh. We thank the Governments
of Bangladesh, Canada, Sweden, and the UK for providing core or
unrestricted support. We thank Jocalyn Clark for superb editorial
assistance.
References
1 Joo I. Cholera vaccines. In: Barua D, Burrows W, eds. Cholera.
Philadelphia, PA: Saunders; 1974: 333–49.
2 Pollitzer R. Cholera. Geneva: World Health Organization, 1959.
3 Harris JB, LaRocque RC, Qadri F, Ryan ET, Calderwood SB.
Cholera. Lancet 2012; 379: 2466–76.
4 Van Heyningen WE, Seal JR. Cholera: The American scientific
experience 1947–1980. Boulder, CO: Westview Press, 1983.
5 Snow J. On the mode of communication of cholera. London:
John Churchill, 1855.
6 Koch R. Der zweite Bericht der deutschen cholera-commission.
Dtsch Med Wochenschr 1883; 9: 743.
7 Baine WB, Mazzotti M, Greco D, et al. Epidemiology of cholera in
Italy in 1973. Lancet 1974; 2: 1370–74.
8 Cvjetanovic B, Barua D. The seventh pandemic of cholera.
Nature 1972; 239: 137–38.
9 British Medical Journal. Cholera in Spain. BMJ 1971; 3: 266.
10 Goodgame RW, Greenough WB. Cholera in Africa: a message for
the West. Ann Intern Med 1975; 82: 101–06.
11 Kustner HG, Gibson IH, Carmichael TR, et al. The spread of
cholera in South Africa. S Afr Med J 1981; 60: 87–90.
12 Swerdlow DL, Mintz ED, Rodriguez M, et al. Waterborne
transmission of epidemic cholera in Trujillo, Peru: lessons for a
continent at risk. Lancet 1992; 340: 28–33.

13 Tanamal ST. Notes on paracholera in Sulawesi (Celebes).
Am J Trop Med Hyg 1959; 8: 72–78.
14 Weil O, Berche P. The cholera epidemic in Ecuador: towards an
endemic in Latin America. Rev Epidemiol Sante Publique 1992;
40: 145–55.
15 Ali M, Nelson AR, Lopez AL, Sack DA. Updated global burden of
cholera in endemic countries. PLoS Negl Trop Dis 2015;
9: e0003832.
16 Colwell RR. Global climate and infectious disease: the cholera
paradigm. Science 1996; 274: 2025–31.
17 Kirn TJ, Jude BA, Taylor RK. A colonization factor links
Vibrio cholerae environmental survival and human infection. Nature
2005; 438: 863–66.
18 Morris JG, Jr. Cholera and other types of vibriosis: a story of human
pandemics and oysters on the half shell. Clin Infect Dis 2003;
37: 272–80.
19 Sack DA, Sack RB, Nair GB, Siddique AK. Cholera. Lancet 2004;
363: 223–33.
20 Meibom KL, Blokesch M, Dolganov NA, Wu CY, Schoolnik GK.
Chitin induces natural competence in Vibrio cholerae. Science 2005;
310: 1824–27.
21 Waldor MK, Mekalanos JJ. Lysogenic conversion by a filamentous
phage encoding cholera toxin. Science 1996; 272: 1910–14.
22 Devault AM, Golding GB, Waglechner N, et al. Second-pandemic
strain of Vibrio cholerae from the Philadelphia cholera outbreak of
1849. N Engl J Med 2014; 370: 334–40.
23 Cholera Working Group. Large epidemic of cholera-like disease in
Bangladesh caused by Vibrio cholerae O139 synonym Bengal. Lancet
1993; 342: 387–90.
24 Johnson JA, Salles CA, Panigrahi P, et al. Vibrio cholerae O139
synonym bengal is closely related to Vibrio cholerae El Tor but has
important differences. Infect Immun 1994; 62: 2108–10.
25 Cameron DN, Khambaty FM, Wachsmuth IK, Tauxe RV,
Barrett TJ. Molecular characterization of Vibrio cholerae O1 strains
by pulsed-field gel electrophoresis. J Clin Microbiol 1994;
32: 1685–90.
26 Safa A, Sultana J, Dac Cam P, Mwansa JC, Kong RY. Vibrio cholerae
O1 hybrid El Tor strains, Asia and Africa. Emerg Infect Dis 2008;
14: 987–88.
27 Chin CS, Sorenson J, Harris JB, et al. The origin of the Haitian
cholera outbreak strain. N Engl J Med 2011; 364: 33–42.
28 Siddique AK, Nair GB, Alam M, et al. El Tor cholera with severe
disease: a new threat to Asia and beyond. Epidemiol Infect 2010;
138: 347–52.
29 World Health Organization. Cholera vaccines: WHO position paper.
Wkly Epidemiol Rec 2010; 85: 117–28.
30 Clemens J, Sack D, Rao M, et al. The design and analysis of cholera
vaccine trials: recent lessons from Bangladesh. Int J Epidemiol 1993;
22: 724–30.
31 Hornick RB, Music SI, Wenzel R, et al. The Broad Street pump
revisited: response of volunteers to ingested cholera vibrios.
Bull N Y Acad Med 1971; 47: 1181–91.
32 Glass RI, Lee JV, Huq MI, Hossain KM, Khan MR. Phage types of
Vibrio cholerae O1 biotype El Tor isolated from patients and family
contacts in Bangladesh: epidemiologic implications. J Infect Dis
1983; 148: 998–1004.
33 Faruque SM, Biswas K, Udden SM, et al. Transmissibility of
cholera: in vivo-formed biofilms and their relationship to infectivity
and persistence in the environment. Proc Natl Acad Sci USA 2006;
103: 6350–55.
34 Nelson EJ, Chowdhury A, Harris JB, et al. Complexity of rice-water
stool from patients with Vibrio cholerae plays a role in the
transmission of infectious diarrhea. Proc Natl Acad Sci USA 2007;
104: 19091–96.
35 Weil AA, Begum Y, Chowdhury F, et al. Bacterial shedding in
household contacts of cholera patients in Dhaka, Bangladesh.
Am J Trop Med Hyg 2014; 91: 738–42.
36 Hartley DM, Morris JG, Jr., Smith DL. Hyperinfectivity: a critical
element in the ability of V. cholerae to cause epidemics? PLoS Med
2006; 3: e7.
37 Jutla AS, Akanda AS, Griffiths JK, Colwell R, Islam S. Warming
oceans, phytoplankton, and river discharge: implications for cholera
outbreaks. Am J Trop Med Hyg 2011; 85: 303–08.
www.thelancet.com Vol 390 September 23, 2017 1547
Downloaded for Anonymous User (n/a) at Riga Stradins University from ClinicalKey.com by Elsevier on October 12, 2018.
For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved.
Seminar
38 Koelle K, Rodo X, Pascual M, Yunus M, Mostafa G.
Refractory periods and climate forcing in cholera dynamics. Nature
2005; 436: 696–700.
39 Pascual M, Rodo X, Ellner SP, Colwell R, Bouma MJ.
Cholera dynamics and El Nino-Southern Oscillation. Science 2000;
289: 1766–69.
40 Faruque SM. Role of phages in the epidemiology of cholera.
Curr Top Microbiol Immunol 2014; 379: 165–80.
41 Mutreja A, Kim DW, Thomson NR, et al. Evidence for multiple
waves of global transmission within the seventh cholera pandemic.
Nature 2011; 477: 462–65.
42 Waldor MK, Tschape H, Mekalanos JJ. A new type of conjugative
transposon encodes resistance to sulfamethoxazole, trimethoprim,
and streptomycin in Vibrio cholerae O139. J Bacteriol 1996;
178: 4157–65.
43 Clemens J, Shin S, Sur D, Nair GB, Holmgren J. New-generation
vaccines against cholera. Nat Rev Gastroenterol Hepatol 2011;
8: 701–10.
44 Banwell JG, Pierce NF, Mitra RC, et al. Intestinal fluid and
electrolyte transport in human cholera. J Clin Invest 1970;
49: 183–95.
45 Carpenter CC, Sack RB, Feeley JC, Steenberg RW. Site and
characteristics of electrolyte loss and effect of intraluminal glucose
in experimental canine cholera. J Clin Invest 1968; 47: 1210–20.
46 De SN. Enterotoxicity of bacteria-free culture-filtrate of
Vibrio cholerae. Nature 1959; 183: 1533–34.
47 Lonnroth I, Holmgren J. Subunit structure of cholera toxin.
J Gen Microbiol 1973; 76: 417–27.
48 Holmgren J, Lonnroth I, Svennerholm L. Tissue receptor for
cholera exotoxin: postulated structure from studies with GM1
ganglioside and related glycolipids. Infect Immun 1973; 8: 208–14.
49 Field M, Fromm D, al-Awqati Q, Greenough WB 3rd. Effect of
cholera enterotoxin on ion transport across isolated ileal mucosa.
J Clin Invest 1972; 51: 796–804.
50 Gill DM, Meren R. ADP-ribosylation of membrane proteins
catalyzed by cholera toxin: basis of the activation of adenylate
cyclase. Proc Natl Acad Sci USA 1978; 75: 3050–54.
51 Holmgren J. Actions of cholera toxin and the prevention and
treatment of cholera. Nature 1981; 292: 413–17.
52 Ouchterlony O, Holmgren J. Cholera and related diarrheas:
molecular aspects of a global health problem; 43rd Nobel
Symposium, Stockholm, August 6–11, 1978. Stockholm: Karger,
1978.
53 Hardy SJ, Holmgren J, Johansson S, Sanchez J, Hirst TR.
Coordinated assembly of multisubunit proteins: oligomerization of
bacterial enterotoxins in vivo and in vitro. Proc Natl Acad Sci USA
1988; 85: 7109–13.
54 Wernick NL, Chinnapen DJ, Cho JA, Lencer WI. Cholera toxin:
an intracellular journey into the cytosol by way of the endoplasmic
reticulum. Toxins 2010; 2: 310–25.
55 Tsai B, Rapoport TA. Unfolded cholera toxin is transferred to the ER
membrane and released from protein disulfide isomerase upon
oxidation by Ero1. J Cell Biol 2002; 159: 207–16.
56 Gabriel SE, Brigman KN, Koller BH, Boucher RC, Stutts MJ. Cystic
fibrosis heterozygote resistance to cholera toxin in the cystic fibrosis
mouse model. Science 1994; 266: 107–09.
57 Lundgren O. Enteric nerves and diarrhoea. Pharmacol Toxicol 2002;
90: 109–20.
58 Herrington DA, Hall RH, Losonsky G, Mekalanos JJ, Taylor RK,
Levine MM. Toxin, toxin-coregulated pili, and the toxR regulon are
essential for Vibrio cholerae pathogenesis in humans. J Exp Med
1988; 168: 1487–92.
59 Taylor RK, Miller VL, Furlong DB, Mekalanos JJ. Use of phoA gene
fusions to identify a pilus colonization factor coordinately regulated
with cholera toxin. Proc Natl Acad Sci USA 1987; 84: 2833–37.
60 Silva AJ, Leitch GJ, Camilli A, Benitez JA. Contribution of
hemagglutinin/protease and motility to the pathogenesis of El Tor
biotype cholera. Infect Immun 2006; 74: 2072–79.
61 Butler SM, Camilli A. Going against the grain: chemotaxis and
infection in Vibrio cholerae. Nat Rev Microbiol 2005; 3: 611–20.
62 Sanchez J, Holmgren J. Cholera toxin structure, gene regulation
and pathophysiological and immunological aspects. Cell Mol Life Sci
2008; 65: b1347–60.
 Seminar
63 Rothenbacher FP, Zhu J. Efficient responses to host and bacterial
signals during Vibrio cholerae colonization. Gut Microbes 2014;
5: 120–28.
64 Evans CA, Gilman RH, Rabbani GH, Salazar G, Ali A. Gastric acid
secretion and enteric infection in Bangladesh.
Trans R Soc Trop Med Hyg 1997; 91: 681–85.
65 Harris JB, Khan AI, LaRocque RC, et al. Blood group, immunity,
and risk of infection with Vibrio cholerae in an area of endemicity.
Infect Immun 2005; 73: 7422–27.
66 Glass RI, Holmgren J, Haley CE, et al. Predisposition for cholera of
individuals with O blood group. Possible evolutionary significance.
Am J Epidemiol 1985; 121: 791–96.
67 Clemens JD, Sack DA, Harris JR, et al. ABO blood groups and
cholera: new observations on specificity of risk and modification of
vaccine efficacy. J Infect Dis 1989; 159: 770–73.
68 Flach CF, Qadri F, Bhuiyan TR, et al. Broad up-regulation of innate
defense factors during acute cholera. Infect Immun 2007;
75: 2343–50.
69 Larocque RC, Sabeti P, Duggal P, et al. A variant in long palate,
lung and nasal epithelium clone 1 is associated with cholera in a
Bangladeshi population. Genes Immun 2009; 10: 267–72.
70 Karlsson EK, Harris JB, Tabrizi S, et al. Natural selection in a
Bangladeshi population from the cholera-endemic ganges river
delta. Sci Transl Med 2013; 5: 192–86.
71 Clemens JD, Sack DA, Harris JR, et al. Breast feeding and the risk
of severe cholera in rural Bangladeshi children. Am J Epidemiol
1990; 131: 400–11.
72 Harris JB, LaRocque RC, Chowdhury F, et al. Susceptibility to
Vibrio cholerae infection in a cohort of household contacts of patients
with cholera in Bangladesh. PLoS Negl Trop Dis 2008; 2: e221.
73 Hsiao A, Ahmed AM, Subramanian S, et al. Members of the
human gut microbiota involved in recovery from Vibrio cholerae
infection. Nature 2014; 515: 423–26.
74 von Seidlein L, Wang XY, Macuamule A, et al. Is HIV infection
associated with an increased risk for cholera? Findings from a
case-control study in Mozambique. Trop Med Int Health 2008;
13: 683–88.
75 Ali M, Emch M, Park JK, Yunus M, Clemens J. Natural cholera
infection-derived immunity in an endemic setting. J Infect Dis 2011;
204: 912–18.
76 Bhuiyan TR, Hoq MR, Nishat NS, et al. Enumeration of gut-homing
β7-positive, pathogen-specific antibody-secreting cells in whole
blood from enterotoxigenic Escherichia coli- and Vibrio cholerae-
infected patients, determined using an enzyme-linked
immunosorbent spot assay technique. Clin Vaccine Immunol 2015;
23: 27–36.
77 Bhuiyan TR, Lundin SB, Khan AI, et al. Cholera caused by
Vibrio cholerae O1 induces T-cell responses in the circulation.
Infect Immun 2009; 77: 1888–93.
78 Glass RI, Svennerholm AM, Khan MR, Huda S, Huq MI,
Holmgren J. Seroepidemiological studies of El Tor cholera in
Bangladesh: association of serum antibody levels with protection.
J Infect Dis 1985; 151: 236–42.
79 Leung DT, Rahman MA, Mohasin M, et al. Comparison of memory
B cell, antibody-secreting cell, and plasma antibody responses in
young children, older children, and adults with infection caused by
Vibrio cholerae O1 El Tor Ogawa in Bangladesh.
Clin Vaccine Immunol 2011; 18: 1317–25.
80 Svennerholm AM, Jertborn M, Gothefors L, Karim AM, Sack DA,
Holmgren J. Mucosal antitoxic and antibacterial immunity after
cholera disease and after immunization with a combined
B subunit-whole cell vaccine. J Infect Dis 1984; 149: 884–93.
81 Uddin T, Harris JB, Bhuiyan TR, et al. Mucosal immunologic
responses in cholera patients in Bangladesh. Clin Vaccine Immunol
2011; 18: 506–12.
82 Clemens JD, van Loon F, Sack DA, et al. Field trial of oral cholera
vaccines in Bangladesh: serum vibriocidal and antitoxic
antibodies as markers of the risk of cholera. J Infect Dis 1991;
163: 1235–42.
83 Holmgren J, Svennerholm AM. Cholera and the immune response.
Prog Allergy 1983; 33: 106–19.
84 Leung DT, Chowdhury F, Calderwood SB, Qadri F, Ryan ET.
Immune responses to cholera in children.
Expert Rev Anti Infect Ther 2012; 10: 435–44.
1548 www.thelancet.com Vol 390 September 23, 2017
Downloaded for Anonymous User (n/a) at Riga Stradins University from ClinicalKey.com by Elsevier on October 12, 2018.
For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved.
85 Jonson G, Osek J, Svennerholm AM, Holmgren J. Immune
mechanisms and protective antigens of Vibrio cholerae serogroup
O139 as a basis for vaccine development. Infect Immun 1996;
64: 3778–85.
86 Clemens JD, Sack DA, Harris JR, et al. Field trial of oral cholera
vaccines in Bangladesh. Lancet 1986; 2: 124–27.
87 Glass RI, Svennerholm AM, Stoll BJ, et al. Protection against
cholera in breast-fed children by antibodies in breast milk.
N Engl J Med 1983; 308: 1389–92.
88 Lange S, Holmgren J. Protective antitoxic cholera immunity in
mice: influence of route and number of immunizations and mode
of action of protective antibodies. Acta Pathol Microbiol Scand C
1978; 86c: 145–52.
89 Centers for Disease Control and Prevention. Laboratory
identification of Vibrio cholerae. 2016. http://www.cdc.gov/cholera/
pdf/laboratory-methods-for-the-diagnosis-of-vibrio-cholerae-
chapter-6.pdf (accessed Sept 11, 2016).
90 Telesmanich NR, Lomov Iu M. Laboratory diagnosis of cholera:
analysis and prospects for improvement. Klin Lab Diagn 2009;
11: 51–55 (in Russian).
91 Benenson AS, Islam MR, Greenough WB 3rd. Rapid identification
of Vibrio cholerae by darkfield microscopy. Bull World Health Organ
1964; 30: 827–31.
92 Bhuiyan NA, Qadri F, Faruque AS, et al. Use of dipsticks for rapid
diagnosis of cholera caused by Vibrio cholerae O1 and O139 from
rectal swabs. J Clin Microbiol 2003; 41: 3939–41.
93 George CM, Rashid MU, Sack DA, et al. Evaluation of enrichment
method for the detection of Vibrio cholerae O1 using a rapid dipstick
test in Bangladesh. Trop Med Int Health 2014; 19: 301–07.
94 Qadri F, Hasan JA, Hossain J, et al. Evaluation of the monoclonal
antibody-based kit Bengal SMART for rapid detection of
Vibrio cholerae O139 synonym Bengal in stool samples.
J Clin Microbiol 1995; 33: 732–34.
95 Rahman M, Sack DA, Mahmood S, Hossain A. Rapid diagnosis of
cholera by coagglutination test using 4-h fecal enrichment cultures.
J Clin Microbiol 1987; 25: 2204–06.
96 Ramamurthy T, Nair G, Quilici M. Cholera surveillance, rapid
diagnostics and laboratory networks. Wkly Epidemiol Rec 2015;
90: 537–39.
97 Hoshino K, Yamasaki S, Mukhopadhyay AK, et al. Development
and evaluation of a multiplex PCR assay for rapid detection of
toxigenic Vibrio cholerae O1 and O139.
FEMS Immunol Med Microbiol 1998; 20: 201–07.
98 Huang J, Zhu Y, Wen H, et al. Quadruplex real-time PCR assay for
detection and identification of Vibrio cholerae O1 and O139 strains
and determination of their toxigenic potential.
Appl Environ Microbiol 2009; 75: 6981–85.
99 Liu J, Gratz J, Amour C, et al. A laboratory-developed TaqMan Array
Card for simultaneous detection of 19 enteropathogens.
J Clin Microbiol 2013; 51: 472–80.
100 Lindenbaum J, Greenough WB, Islam MR. Antibiotic therapy of
cholera in children. Bull World Health Organ 1967; 37: 529–38.
101 Clemens J, Shin S, Shah B, Sack D. Cholera vaccines. In: Plotkin S,
Orenstein W, Offit P, eds. Vaccines, 5th ed. Amsterdam: Elsevier, 2013.
102 Tariq M, Memon M, Jafferani A, et al. Massive fluid requirements
and an unusual BUN/creatinine ratio for pre-renal failure in
patients with cholera. PLoS One 2009; 4: e7552.
103 Bennish ML, Azad AK, Rahman O, Phillips RE. Hypoglycemia
during diarrhea in childhood. Prevalence, pathophysiology, and
outcome. N Engl J Med 1990; 322: 1357–63.
104 Ciglenecki I, Bichet M, Tena J, et al. Cholera in pregnancy:
outcomes from a specialized cholera treatment unit for pregnant
women in Leogane, Haiti. PLoS Negl Trop Dis 2013; 7: e2368.
105 Alam NH, Ashraf H. Treatment of infectious diarrhea in children.
Paediatr Drugs 2003; 5: 151–65.
106 WHO. Pocket book of hospital care for children: guidelines for the
management of common childhood illnesses, 2nd edn. Geneva:
World Health Organization, 2013.
107 WHO. The treatment of diarrhoea: a manual for physicians and
other senior health workers (4th revision). Geneva: World Health
Organization, 2005.
108 Ramakrishna BS, Venkataraman S, Srinivasan P, Dash P,
Young GP, Binder HJ. Amylase-resistant starch plus oral
rehydration solution for cholera. N Engl J Med 2000; 342: 308–13.

109 Ahmed T, Ali M, Ullah MM, et al. Mortality in severely
malnourished children with diarrhoea and use of a standardised
management protocol. Lancet 1999; 353: 1919–22.
110 WHO Multicentre Growth Reference Study Group. WHO child
growth standards based on length/height, weight and age.
Acta Paediatr Suppl 2006; 450: 76–85.
111 WHO. Guideline: updates on the management of severe acute
malnutrition in infants and children. Geneva: World Health
Organization, 2013.
112 Alam NH, Hamadani JD, Dewan N, Fuchs GJ. Efficacy and safety of
a modified oral rehydration solution (ReSoMaL) in the treatment of
severely malnourished children with watery diarrhea. J Pediatr
2003; 143: 614–19.
113 Greenough WB 3rd, Gordon RS Jr, Rosenberg IS, Davies BI,
Benenson AS. Tetracycline in the treatment of cholera. Lancet 1964;
1: 355–57.
114 Kaushik JS, Gupta P, Faridi MM, Das S. Single dose azithromycin
versus ciprofloxacin for cholera in children: a randomized
controlled trial. Indian Pediatr 2010; 47: 309–15.
115 Saha D, Karim MM, Khan WA, Ahmed S, Salam MA, Bennish ML.
Single-dose azithromycin for the treatment of cholera in adults.
N Engl J Med 2006; 354: 2452–62.
116 Roy SK, Hossain MJ, Khatun W, et al. Zinc supplementation in
children with cholera in Bangladesh: randomised controlled trial.
BMJ 2008; 336: 266–68.
117 WHO. Clinical management of acute diarrhoea. WHO/FCH/
CAH/04.7. Geneva: World Health Organization/Unicef joint
statement, 2004.
118 Cutler D, Miller G. The role of public health improvements in
health advances: the twentieth-century United States. Demography
2005; 42: 1–22.
119 WHO, UNICEF. Progress on sanitation and drinking water: 2015
update and MDG assessment. Geneva: World Health Organization,
Unicef, 2015.
120 Colwell RR, Huq A, Islam MS, et al. Reduction of cholera in
Bangladeshi villages by simple filtration. Proc Natl Acad Sci USA
2003; 100: 1051–55.
121 Deb BC, Sircar BK, Sengupta PG, et al. Studies on interventions to
prevent El Tor cholera transmission in urban slums.
Bull World Health Organ 1986; 64: 127–31.
122 O’Connor KA, Cartwright E, Loharikar A, et al. Risk factors early in
the 2010 cholera epidemic, Haiti. Emerg Infect Dis 2011; 17: 2136–38.
123 George CM, Monira S, Sack DA, et al. Randomized controlled trial
of hospital-based hygiene and water treatment intervention
(CHoBI7) to reduce cholera. Emerg Infect Dis 2016; 22: 233–41.
124 Benenson AS, Joseph PR, Oseasohn RO. Cholera vaccine field trials
in east Pakistan. 1. Reaction and antigenicity studies.
Bull World Health Organ 1968; 38: 347–57.
125 Cash RA, Music SI, Libonati JP, Craig JP, Pierce NF, Hornick RB.
Response of man to infection with Vibrio cholerae. II. Protection
from illness afforded by previous disease and vaccine. J Infect Dis
1974; 130: 325–33.
126 Cohen MB, Giannella RA, Bean J, et al. Randomized, controlled
human challenge study of the safety, immunogenicity, and
protective efficacy of a single dose of Peru-15, a live attenuated oral
cholera vaccine. Infect Immun 2002; 70: 1965–70.
127 Coster TS, Killeen KP, Waldor MK, et al. Safety, immunogenicity,
and efficacy of live attenuated Vibrio cholerae O139 vaccine
prototype. Lancet 1995; 345: 949–52.
128 Garcia L, Jidy MD, Garcia H, et al. The vaccine candidate
Vibrio cholerae 638 is protective against cholera in healthy
volunteers. Infect Immun 2005; 73: 3018–24.
www.thelancet.com Vol 390 September 23, 2017 1549
Downloaded for Anonymous User (n/a) at Riga Stradins University from ClinicalKey.com by Elsevier on October 12, 2018.
For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved.
Seminar
129 Levine MM, Kaper JB, Herrington D, et al. Safety, immunogenicity,
and efficacy of recombinant live oral cholera vaccines, CVD 103 and
CVD 103-HgR. Lancet 1988; 2: 467–70.
130 Liang W, Wang S, Yu F, et al. Construction and evaluation of a safe,
live, oral Vibrio cholerae vaccine candidate, IEM108. Infect Immun
2003; 71: 5498–504.
131 Mahalanabis D, Ramamurthy T, Nair GB, et al. Randomized
placebo controlled human volunteer trial of a live oral cholera
vaccine VA1.3 for safety and immune response. Vaccine 2009;
27: 4850–56.
132 Tacket CO, Losonsky G, Nataro JP, et al. Initial clinical studies of
CVD 112 Vibrio cholerae O139 live oral vaccine: safety and efficacy
against experimental challenge. J Infect Dis 1995; 172: 883–86.
133 Richie EE, Punjabi NH, Sidharta YY, et al. Efficacy trial of
single-dose live oral cholera vaccine CVD 103-HgR in North Jakarta,
Indonesia, a cholera-endemic area. Vaccine 2000; 18: 2399–410.
134 Chen WH, Cohen MB, Kirkpatrick BD, et al. Single-dose live oral
cholera vaccine CVD 103-HgR protects against human experimental
infection with Vibrio cholerae O1 El Tor. Clin Infect Dis 2016;
62: 1329–35.
135 Lewis DJ, Gilks CF, Ojoo S, et al. Immune response following oral
administration of cholera toxin B subunit to HIV-1-infected UK and
Kenyan subjects. AIDS 1994; 8: 779–85.
136 Bhattacharya SK, Sur D, Ali M, et al. 5 year efficacy of a bivalent
killed whole-cell oral cholera vaccine in Kolkata, India: a cluster-
randomised, double-blind, placebo-controlled trial. Lancet Infect Dis
2013; 13: 1050–56.
137 Qadri F, Wierzba TF, Ali M, et al. Efficacy of a single-dose,
inactivated oral cholera vaccine in Bangladesh. N Engl J Med 2016;
374: 1723–32.
138 WHO. Potential use of oral cholera vaccines in emergency
situations: report of a WHO meeting, Geneva, Switzerland,
12–13 May, 1999. http://apps.who.int/iris/bitstream/10665/66004/1/
WHO_CDS_CSR_EDC_99.4.pdf (accessed Feb 20, 2017).
139 WHO. Cholera, 2014. Wkly Epidemiol Rec 2015; 90: 517–44.
140 Harris JB, Larocque RC, Charles RC, Mazumder RN, Khan AI,
Bardhan PK. Cholera’s western front. Lancet 2010; 376: 1961–55.
141 Ali M, Emch M, von Seidlein L, et al. Herd immunity conferred by
killed oral cholera vaccines in Bangladesh: a reanalysis. Lancet 2005;
366: 44–49.
142 Longini IM, Jr., Nizam A, Ali M, Yunus M, Shenvi N, Clemens JD.
Controlling endemic cholera with oral vaccines. PLoS Med 2007;
4: e336.
143 WHO. WHO Technical Working Group on creation of an oral
cholera vaccine stockpile: meeting report, Geneva, 26–27 April,
2012. http://apps.who.int/iris/bitstream/10665/75240/1/WHO_
HSE_PED_2012_2_eng.pdf (accessed Feb 20, 2017).
144 Luquero FJ, Grout L, Ciglenecki I, et al. Use of Vibrio cholerae
vaccine in an outbreak in Guinea. N Engl J Med 2014; 370: 2111–20.
145 Martin S, Lopez AL, Bellos A, et al. Post-licensure deployment of
oral cholera vaccines: a systematic review. Bull World Health Organ
2014; 92: 881–93.
146 Qadri F, Ali M, Chowdhury F, et al. Feasibility and effectiveness of
oral cholera vaccine in an urban endemic setting in Bangladesh:
a cluster randomised open-label trial. Lancet 2015; 386: 1362–71.
147 Clemens J, Holmgren J. When, how, and where can oral cholera
vaccines be used to interrupt cholera outbreaks?
Curr Top Microbiol Immunol 2014; 379: 231–58.