Case study

An Outbreak of Cholera

West Bengal, India

This case study was developed by a working group led by the World Health Organization (WHO)
EPR with cooperation from the Field Epidemiology Training Programme (FETP) from the National
Institute of Epidemiology (NIE), Indian Council of Medical Research (ICMR), Chennai, Tamil Nadu,
India.

It is inspired by an investigation conducted by Dr Tapas Kumar Sen, 2003 MAE scholar assigned
to the district of 24 Parganas, West Bengal, India..
 Using this case study in the classroom:
We recommend that this case study be used in conjunction with the “Laboratory Skills for
Epidemiologists” module, developed by the WHO. However, it can be delivered on its own,
provided key lectures are presented first.

Recommended prerequisite lectures (cf. training matrix):

Lecture 3: Taking appropriate and adequate samples safely.
Lecture 4: Transport, disinfection and biosafety
Lecture 15: Anti-microbial susceptibility testing
Lecture 16: Role of the laboratory in surveillance
Lecture 17: Quality assurance

Time required for this case study:

3hours

This case study does not come with a facilitator’s guide. The answers to all the questions for each
section are provided as an introduction to the following section.

To run this case study in the classroom, we propose that it be distributed one page at a time.

Participants should take turns reading it aloud, paragraph by paragraph. Reading all paragraphs
aloud and in turns has two advantages: first, everyone can quickly participate and get beyond the
inhibition of having her/his voice heard in a large room; second, time is given to the whole class to
understand the issue and think about the answers. The participants reading the question may try to
answer it if s/he can; otherwise, it can be discussed as a group. The next participant reads the next
question and so on until the end of the page. After the next part/page is distributed, the next
participant continues and so on until the case study is over. Once the epilogue is read, the class
should re-visit the objectives, which reinforces their learning and provides an opportunity to clarify
what may not have been fully understood.

Learning objectives
At the end of the case study, the student will be able to:
1. Understand the role of the laboratory in surveillance and outbreak detection;
2. Identify the number of samples to take and the transport media to use during an outbreak of
suspected cholera;
3. Provide the minimum information needed to ensure appropriate laboratory testing;
4. Take appropriate measures to prevent contamination and/or infection when collecting
specimens to send to a laboratory;
5. Identify the appropriate laboratory tests to request during an outbreak of diarrhea;
6. Understand the key elements of quality assurance for laboratories;
7. Interpret Vibrio cholerae biotypes and serotypes;
8. Understand the relevance of antibiotic susceptibility testing during the investigation of an
outbreak of cholera;
9. Decide whether environmental samples are needed during an outbreak of diarrhea;
10. Integrate epidemiological and laboratory data to formulate conclusions during cholera outbreak
investigations.

Cholera India, May 2007 2

Part 1. A visit to the National Institute of Cholera and Enteric Diseases,
West Bengal, India
The National Institute of Cholera and Enteric Diseases (NCED) is a specialized Institute of the
Indian Council for Medical Research (ICMR) located in Kolkota, West Bengal, India. Cholera is
highly endemic in this region of India so while the NCED works as a reference centre for the entire
country, the institute is mainly active in West Bengal.

Cholera is an infectious, acute watery diarrhea caused by Vibrio cholerae O1 and O139. Vibrio
cholerae produce a powerful toxin that stimulates the secretion of water and electrolytes in the
intestinal tract. Patients with cholera commonly suffer from acute dehydration.1 In India, Vibrio
cholerae O139 emerged since 1992.

In October 2004, the epidemiologist assigned to “North 24 Parganas” (a district in West Bengal)
conducted a routine visit to the NCED and its laboratory. The microbiologist in charge of cholera
mentioned to him that during the previous month (September), the laboratory isolated Vibrio
cholerae from 65 stool samples, a substantial increase over the average isolation of Vibrio
cholerae from 19 stool specimens each month between January and August, 2004 (Table 1).

Table 1: Number of stool samples from which Vibrio cholerae was isolated, National Centre
for Cholera and Diarrheal Diseases, Kolkota, West Bengal, India 2004 1
Jan Feb Mar Apr May Jun Jul Aug Sep
Number of stool samples
from which Vibrio cholerae 0 2 5 20 12 10 7 15 65
was isolated

Question 1a
What is the role of the laboratory in public health surveillance?

Question 1b
How can these data be interpreted? Is this an outbreak?

1
Fictional data

Cholera India, May 2007 3

Discussion for Part 1

Laboratories play a very important role in public health surveillance when they work in collaboration
with epidemiologists. For example, case definitions which include laboratory criteria are more
specific – but to be able to classify someone based on laboratory confirmation, epidemiologists
must collaborate with a network of laboratories to ensure prompt testing and reporting of
specimens, particularly during investigations.

With laboratory results reported back to public health in a timely way, epidemiologists can then
calculate incidence rates: the number of cases confirmed by the laboratory would be the
numerator, and the population at risk, the denominator. For example, for poliomyelitis, health care
facilities notify public health authorities when they diagnose acute flaccid paralysis, and stool
samples are then sent to laboratories. When the laboratory results are reported back, surveillance
officers can calculate incidence. Thus, when laboratory data are linked with epidemiological data,
the quality of public health surveillance improves.

The laboratory may also take a lead role in surveillance of specific conditions for which clinical
criteria are insufficient. Some countries will use laboratory-based surveillance for hepatitis A or
acute hepatitis B, where identifying positive tests (IgM antibodies to hepatitis A virus or IgM
antibodies to hepatitis B virus core antigen) may be more specific or meaningful than looking for
cases of acute jaundice. In other cases, laboratories can play a lead role in surveillance through
the identification of clusters of unusual infections for which reference laboratories may centralize
biological samples (e.g., Global SalmSurv, the global surveillance system for Salmonella that uses
serotyping).

In India, public health surveillance is mostly based on notification of clinical syndromes, for which
laboratory confirmation is sometimes needed. The keystone of cholera surveillance is the
surveillance for acute watery diarrhea1 occurring as an isolated case or in clusters. Laboratory
confirmation is sought for clinical cases of cholera. Since cholera surveillance is not solely
laboratory-based, it is difficult to interpret the data in Table 1 in the absence of more information.

An outbreak is defined as an increase in incidence, but calculating incidence requires both a

numerator and denominator. Table 1 includes only numerator data.

In order to interpret the data in Table 1, it is important to know the reasons behind sample
collection and testing, as well as the total number of samples tested. Since West Bengal is
endemic for cholera and faces seasonal recrudescence in September and October, it may be that
the observed increase reflects several separate, localized outbreaks that should be investigated
individually.

Part 2. Initiation of an epidemiological investigation

On 13 October 2004, with the epidemiologist back in his district office, a health care facility in
Kanchrapara municipality reports a cluster of cases of acute watery diarrhea with a high proportion
of cases admitted to hospital with acute dehydration. The cluster is suspected to be an outbreak of
cholera.

Question 2a
What are the initial steps of the epidemiological field investigation?

Question 2b
Should a microbiologist be part of the investigation team in the field?

1
Diarrhea is defined as at least three loose stools in a 24-hour period.

Cholera India, May 2007 4

Discussion for Part 2

A first step of any investigation is to confirm the existence of the outbreak. In this situation, the
epidemiologist reviewed available data from Kanchrapara municipality, for the number of (severe)
acute watery diarrhea reported each month – which was 25 on average during the first nine
months of 2004.

Further review of the surveillance data indicated that all patients lived between Kulia Road,
Dharmanagar Colony and Ambagan area, in the “ward” (part of town) four and five of Kanchrapara
Municipality. In the absence of any change in the reporting system and in the absence of any
influx of population, the epidemiologist concluded that the current reports may likely be a true
increase of incidence

The second step of the investigation is to confirm the diagnosis. A short visit to the hospital
verified that patients had signs and symptoms, including dehydration, compatible with cholera. The
epidemiologist decided to obtain laboratory confirmation of the diagnosis.

As a third step of the investigation, epidemiologists define a case. In this example, a case was
defined as a patient aged five years or more, living in Kulia Road-Dharmanagar Colony-Ambagan
area of ward four or five (Kanchrapara Muncipality since 5 October 2004. In developing a case
definition, he referred to the World Health Organization (WHO) case definition in a reference
surveillance document downloaded from the WHO Internet site (www.who.int). 12

While it would be convenient to have a microbiologist each time an epidemiologist conducts an

investigation in the field, microbiologists usually do not participate in the field. However, it is
important to recognize that there will be a need to consult with the laboratory and infectious
disease specialists at an early stage.

Part 3. Collection of laboratory specimens

The epidemiologist is now about to collect specimens for laboratory investigations.

Question 3a
How many case-patients should be sampled? Why? What kind of patients should the
epidemiologist select to take samples?

Question 3b
What kind of infection control measures should be in place to collect the samples?

Question 3c
What kind of samples should be taken? What quantity?

Question 3d
What is a transport medium? When should it be used? What kind of transport media should be
used to take stool specimens in this case?

Question 3e
When is there a need for a cold chain for the transport of specimen? Why?

Question 3f
What other practical issues need to be considered before collecting samples?

1
Age criterion used to increase specificity. It does not mean that cholera does not occur among those under
five years. Culture-proven cases even below one year of age are common in endemic areas.

Cholera India, May 2007 5

Discussion for Part 3

The rule of thumb for the number of samples during a cholera outbreak is 10 samples to confirm
the diagnosis, five samples per week during the outbreak to make sure it is still cholera and to
monitor the antimicrobial resistance pattern and five samples at the end of the outbreak to confirm
that the outbreak is over. Using these criteria, samples were obtained from 10 patients meeting the
case definition for laboratory testing and who had NOT received antibiotics.  It is important to
collect enough samples to avoid sampling error while not collecting too many, which may
overwhelm the laboratory.

After consulting with the microbiologist and the clinician, the epidemiologist is advised to obtain
rectal swabs from a sample of patients. Rectal swabs are small enough to allow easy and safe
transport yet sufficient to yield Vibrio cholerae when it is present (however, they are less adapted
for Salmonella). Before obtaining the specimens, hands should be washed and gloves adorned; a
white lab coat should be worn when collecting biologic specimens. To obtain a proper rectal swab,
a clean cotton tipped swab should be introduced in the rectum until it becomes moist. It is
important to avoid cross contamination between samples during collection and handling. After
completing the procedure, gloves are removed and hands should be washed.

A transport medium is designed to maintain the viability of microorganisms throughout the period of
collection and shipment. Shelf lives of transport media typically range from six months to one year
when stored at room temperature. It is important to check the expiry date. Such media are used
when we anticipate that samples will take more than a few hours to reach a laboratory. In this
specific case, the epidemiologist decided to use a Cary Blair transport medium. This media is
made of agar, distilled water, Sodium Thioglycolate, NaCl and Na2HPO3. These should be stocked
in districts as an emergency preparedness measure.

Cold chain is generally needed for all specimens, with a few exceptions. One of these exceptions
is rectal swabs for cholera culture in Cary Blair medium that can reach the laboratory within 24
hours. In contrast, stool samples for bacterial culture are always transported under cold chain.3 In
this investigation, the specimens will reach the laboratory in Kolkota within 24 hours, so a cold
chain is not being required.

Remember: The accuracy of any test procedure is dependent on the quality of the specimen. The
quality of the specimen is dependent on how and when it was collected, the care given to its
preservation, and how soon it reaches the laboratory.

Part 4. Labeling of specimens

While the epidemiologist prepares to send the rectal swabs to the laboratory, the physician treating
the patients in the hospital suggests that the samples be sent to the School of Tropical Medicine of
Kolkota, a long-standing hospital partner. 1
To prepare the specimens for transport, the epidemiologist’s assistant asks which forms to fill out
to send with the rectal swabs.

Question 4a
What kind of contact should be made with the laboratory prior to sending the samples?

Question 4b
What kind of information should be included with the samples sent to the laboratory to ensure
appropriate testing?

1
If the hospital had a microbiological lab, they should send the culture isolates for confirmation

Cholera India, May 2007 6

Discussion for Part 4

Before sending any specimens, the epidemiologist should call the laboratory at the School of
Tropical Medicine to speak with the microbiologist. The epidemiologist should explain what is
known about the current situation, including providing initial results of the epidemiological
investigation. The laboratory should confirm that they can accept the samples and that there are
no logistical constraints to receive them (e.g., staff available to receive and test the specimens, no
other laboratory priorities (another outbreak) or just before a week end). The epidemiologist should
make note of who he/she has spoken to the laboratory contact name, title, phone number and e-
mail address and the exact location to which the samples should be delivered. The microbiologist
assigns an outbreak number for the specimens to ensure that all specimens received with this
number would be processed and prioritized accordingly; this also facilitates reporting back of
results. The laboratory contact may also want to ensure that someone from the lab would be
available to receive, handle and store samples as they arrive. They also discussed the potential
media interest in this cholera outbreak – so they clarified that the laboratory results would be
reported directly back to the epidemiologist for appropriate interpretation and dissemination to the
designated public health authority, who would subsequently be responsible for broader
communication to the public.

As a follow up to a phone call, it is always best to summarize information discussed on the phone
in written form and to send the agreed-upon protocol, by email if possible, to interested parties (in
this case, the lab and public health authorities).

After ending this phone conversation, the epidemiologist instructs his assistant to complete a form
with the following information for each patient sampled:
1. Type of sample
2. Name (for diagnostic samples, a complete identifier is needed; for research purposes, a
confidential identifier might be more appropriate)
3. Place of residence
4. Signs and symptoms of the patient
5. Date of onset and date of collection
6. Prior antibiotic treatment if any (which in this case, will be none for all samples)
7. Outbreak number

Part 5. Transport of specimens

As the samples are being prepared for the lab, the person responsible for transporting the samples
to the laboratory refuses to take them in his car, as he is afraid to catch cholera.

Question 5a
What kind of package is necessary to prepare these samples for transport?

Question 5b
How should these samples be transported to the laboratory?

Question 5c
What can be said to reassure the person who will carry the samples?

Cholera India, May 2007 7

Discussion for Part 5

These types of specimens require Category B packaging (according to WHO recommendations)

(Figure 1) 4

1. The sample is placed in a primary watertight receptacle with thick walls.

2. This primary receptacle is then placed in a second watertight container (e.g., hard box
made of metal or plastic) and surrounded by absorbent material (e.g., cotton).
This secondary watertight container is then labeled with the specimen data form taped on
its surface, while another copy of the form is sent to the laboratory by fax.
3. The secondary container is then placed in a hard box (e.g., wood, metal, cardboard,
plastic) and surrounded by more absorbent material.
This outer box displays the address of the laboratory and the contact information of the
sender (Note: a biohazard label is not needed for category B specimens).

1
Figure 1: Transport of rectal swabs taken during an outbreak of suspected cholera.

When packaged in this way, the swabs can be transported safely without additional precautions.5

In this case, the person who was asked to transport the samples was informed that all precautions
had been taken and was reassured that there was no risk of infection to him; he agreed to take the
package and he confirmed the delivery date and time.

1
Class 6.50 package reproduced from the WHO Biosafety guide Error: Reference source not found (initially
provided by the International Air Transport Association, IATA).

Cholera India, May 2007 8

After sending the specimens to the lab, the investigation team completes the fourth step of the
investigation with an active, door-to-door case findng.

They identified 194 cases among 1 301 population (attack rate: 15%, almost identical among
males and females, but higher among persons of 60 years of age or older, Table 2). The
distribution of cases over time suggested a persisting, common source outbreak (Figure 2).

Table 2: Distribution of acute watery diarrhea cases by age and sex, Kanchrapara
Municipality, North 24 Parganas, West Bengal, India, 2004 (n=194)
Attack rate
Population Cases % of total cases
(%)
Age in
5-14 327 54 27.8 16.5
years
15-45 748 105 54.1 14.1
46-60 184 27 14.0 14.7
>60 42 8 4.1 19.0
Sex Male 696 105 54.1 15.1
Female 605 89 45.9 14.7

Figure 1: Distribution of cases of acute watery diarrhea by date of onset, Kanchrapara

Municipality, North 24 Parganas District, West Bengal, India, 2004 (n=194)

60

50

40
Number of cases

30

20

10

6t
h
7t
h
8t
h
9t
h th th th th th th th th th th th st
10 11 12 13 14 15 16 17 18 19 20 21
Date of Onset (October 2004)

Cholera India, May 2007 9

Part 6. Decision tree for laboratory testing
The samples reached the laboratory. The microbiologist calls the epidemiologist to confirm he
received the specimens, and thanks him for all the information provided. They now need to discuss
what to look for in the samples.

Question 6a
In general, how does one decide what laboratory tests to conduct when investigating an outbreak
of diarrheal illness?

Question 6b
In this situation, what laboratory tests should be requested to confirm the diagnosis of cholera?

Question 6c
What categories of laboratory techniques can a laboratory use to identify pathogens in a stool
sample? What technique is the laboratory going to use in this case?

Cholera India, May 2007 10

Discussion for Part 6

The criteria used to determine which laboratory tests to request during diarrheal outbreaks include
(1) the clinical characteristics of the case-patients, (2) the macroscopic characteristics of the stool
sample, (3) the epidemiological characteristics of the outbreak and (4) the geographical setting
where the outbreak takes place.

During this outbreak, in view of (1) watery diarrhea with severe dehydration among adults, (2) the
rice water aspect of the stools, (3) the common source rather than person-to-person propagation
and (4) the high endemicity of cholera in West Bengal, the epidemiologist requested a culture for
cholera and other enteropathogens.

Even though cholera seems the most likely diagnosis, only asking for Vibrio cholerae stool culture
would be too restrictive in the absence of preliminary laboratory results. In addition, in the case of
acute diarrhea, the clinical picture and the macroscopic appearance of the stool sample may be
aspecific initially and may only become more typical as the pathogenesis evolves (e.g., rice water
stool secretory diarrhea after the cholera toxin is released in the intestinal lumen, dysentery after
invasion of the epithelium).

The standard protocol for processing of stool samples for common bacterial pathogens in a basic
laboratory covers at least Salmonella, Shigella and Vibrio cholerae (Figure 3).

Finally, diarrhea due to mixed etiology is common and well-documented. Determining the primary
causative agent may be a difficult issue. Thus, selective media for isolation of Vibrio cholerae may
miss out other pathogens.

Potential bacteria to look for include:

 Vibrio cholerae;
 Salmonella spp;
 Salmonella typhi and paratyphi (generally are causes of fever rather than causes of acute
diarrheal disease outbreak);
 Shigella;
 Campylobacter;
 Yersinia;
 Clostridium. 1

Direct, macroscopic examination of the stool sample may suggest an agent (e.g., “rice water”
stools in the absence of blood suggests cholera). In addition, a number of laboratory techniques
can be used to identify pathogens in a stool sample. These include direct examination (e.g., for
microscopic parasites), gram stain (e.g., clostridium), antigen detection (e.g., for rotavirus), cultures
(e.g., for most bacterial pathogens) and nucleic acid detection (e.g., norovirus).

In this outbreak, the laboratory will culture the rectal swabs in alkaline peptone water (APW) and
look for numerous motile bacteria moving like swarms of gnats/shooting stars appearing as gram
negative, comma shaped bacteria after staining. After subcultures, colonies that will agglutinate the
antiserum O1will be identified as Vibrio cholerae O1. 6

1
Different atmospheric conditions.

Cholera India, May 2007 11

Figure 3: Flow chart for processing diarrheal samples in a laboratory

Diarrheal
stool

Direct Enrichment
plating

Alkaline Selenite
Bile salt peptone F broth/
XLD agar agar/ water Gram
TCBS Negative
agar broth
water
Subculture after 6-
8 hours

Bile salt
agar/ XLD agar
TCBS
agar

Overnight incubation at 37 degree C

Examine the plates

Cholera India, May 2007 12

Meanwhile, the epidemiologist was busy in the field – he drew a spot map (Figure 4), illustrating
the distribution of cases by household, in the area of the outbreak. Most cases clustered around
the Kulia road and Dharmanabar colony areas (both supplied with municipal water supply, shown
as MT on the map) while the Ambagan area (supplied with water from the railways water supply
system, shown as RT on the map) was less affected. Neither water source was chlorinated. No
other exposure (e.g., common event, food) seemed to explain this geographical distribution of
cases.

Figure 4: Distribution of acute watery diarrhea cases by place of residence, Kanchrapara

Municipality, North 24 Parganas District, West Bengal, India, 2004

Part 7. Quality assurance for laboratory testing

The epidemiologist discusses his findings with a colleague. The colleague mentions he has never
heard of the laboratory at the Kolkota School of Tropical Medicine and questions whether the
laboratory samples were sent to a “reliable laboratory”.

Question 7a
Can an epidemiologist assess whether a laboratory is reliable?

Question 7b
What are the measures that a laboratory can take to improve the quality of the results and improve
its reliability?

Cholera India, May 2007 13

Discussion for Part 7

To get a sense of the reliability of a laboratory, an epidemiologist may engage the laboratory in a
dialogue and tactfully ask about quality assurance measures in place. He can also enquire about
the type of assays and/or reagents being issued so that their sensitivity and specificity can be
known. In the case of cholera for instance, he can ask the origin of the antiserum used for the
determination of O antigen and for the serotype. He can also take into account a track record of
successful collaborations on outbreak investigations. However, an epidemiologist is not in a
position to assess the reliability of the laboratory or to evaluate its quality assurance procedures, as
this requires a specific expertise.

Quality assurance measures that improve reliability include:

1. Internal quality control processes, for example:
a. the type and quality of assays, reagent and equipment being used;
b. the presence of a trained microbiologist;
c. adequate staffing;
d. the habit of cross-checking results;
e. the use of standard operating procedures, including documenting procedures and
f. the existence of a continuous training programme for staff.
2. External quality assurance, such as participating in an external quality assurance scheme.

As the fifth step of the investigation, epidemiologists generate a hypothesis on the basis of
descriptive results. In this outbreak, most age groups and both sexes are affected; the distribution
of cases over time suggests a persistent common source outbreak. Cases are clustered around
the neighborhood supplied by the municipal water system. Thus, the municipal water system could
be the source of the outbreak.

Part 8. Environmental testing

The epidemiologist enquires about the water system and learns that on 12 October, a leak was
detected on the pipeline of the municipal water supply. He realizes that this leak could have led to
a contamination of the water for two reasons: first, the municipal water supply is intermittent and
there is negative pressure sucking fluids into the pipeline when water is not distributed; second,
there was accumulation of sewage-contaminated water in the area of pipeline following heavy rains
at the time.

On 13 October, the municipal water company repaired the leak and heavily chlorinated the water
that day. Thus, the epidemiologist suspects that the municipal pipeline water is the source of the
outbreak.

Question 8a
At this stage of the epidemiological investigation, can one consider that the source of the outbreak
is identified? Are additional epidemiological investigations needed?

Question 8b
In general, what kind of laboratory tests can be conducted to assess the contamination of drinking
water? What kind of tests could be conducted on the municipal water system in this situation?

Question 8c
As the open sewage was suspected as a potential source of contamination for the municipal water
system, would it be useful to also test samples from the sewage?

Cholera India, May 2007 14

Discussion for Part 8

As the epidemiologist reviewed the results of the descriptive epidemiology for this outbreak, the
leading hypothesis was that the municipal pipeline water had been contaminated by sewage water
because of a leak in the pipeline and that this contamination affected the Kulia road and
Dharmanabar colony areas. The sixth step of an outbreak investigation is hypothesis testing.
The epidemiologist decides to undertake a cohort study to determine if there is an association
between illness and exposure to the suspected water supply.

Two water tests can be used to assess if drinking water is contaminated: first, the total count of
coliforms could be compared with international references 7 to estimate the level of contamination
of the drinking water; second, the drinking water could be tested for the presence of Vibrio
cholerae--however, testing water samples for the presence of Vibrio cholerae requires large
samples of concentration techniques.

In this outbreak, the epidemiologist decided not to test the municipal water supply because of the
heavy chlorination that followed the leak repair. Ideally, it would have been useful to take a water
sample before the chlorine was added. Testing the open sewage would not have been useful
either. The coliform count would have been high and not very informative. The presence of Vibrio
cholerae in the open sewage could also be a consequence of the outbreak and equally non-
informative.

Part 9. Antibiotic susceptibility testing

The laboratory reports back that Vibrio cholerae O1 was isolated from eight out of the ten rectal
swabs submitted. No other pathogens were found. As the epidemiologist shares these results with
the team, the head of the medicine department asks about the antibiotic susceptibility pattern of
the isolated strain.

Question 9a
How can the isolation of Vibrio cholerae O1 be interpreted in these samples? Why is it that other
microorganisms were not found? What additional information is needed to interpret the negative
results?

Question 9b
Antibiotic resistance patterns should be received within 24 hours of the isolation. How will this
information be used?

Question 9c
What is the relevance of antibiotic susceptibility testing in this outbreak? What kind of limitations
should be kept in mind when interpreting antibiotic resistance patterns?

Cholera India, May 2007 15

Discussion for Part 9

The isolation of Vibrio cholerae O1 in the samples confirmed the hypothesis that this was a cholera
outbreak. The absence of other pathogens strengthened the finding that cholera was not a
coincidental finding and that it was the pathogen implicated in the outbreak. However, the reported
absence of other pathogens must be interpreted in light of the pathogens that the laboratory has
looked for.

In general, antibiotic resistance patterns are used to guide treatment and may be useful to monitor
antibiotic susceptibility profile for surveillance purposes. They may also help to type outbreak
strains.

With cholera, the primary treatment is oral or intravenous rehydration. However, when the epidemic
strain is susceptible, WHO recommends treating adults with tetracycline as it reduces the infectivity
of the stools and may decrease the duration of the symptoms. Error: Reference source not found
Children may be given trimethoprim-sufamethoxazole. Error: Reference source not found Other
antibiotics are unnecessary and should not be used. There is a tendency to overuse associations
of large spectrum antibiotics for the management of cholera.

During this outbreak, the epidemiologist asked the laboratory to assess the antibiotic susceptibility
profile of the strain involved to ensure that tetracycline could be used to treat cases. He requested
this profile, fully aware that the accurate results are contingent on the quality assurance of the
testing laboratory.

Part 10. Typing of a strain of cholera during an outbreak

The complete laboratory report is now available. It specifies that eight out of ten samples have
grown Vibrio cholerae O1. The Vibrio produces acetoin and is resistant to polymyxin B indicating
the biotype “El Tor”. The culture agglutinates the Ogawa antiserum indicating a serotype Ogawa.
All isolates are sensitive to tetracycline.

Question 10a
What is the El Tor biotype? What does this result mean?

Question 10b
What is the Ogawa serotype? What does this result mean?

Cholera India, May 2007 16

Discussion for Part 10

The El Tor biotype is responsible for the current pandemic of cholera that started in 1937 in the
Celebes. This biotype causes less severe disease than “classical” cholera. The isolation of the El
Tor biotype in this outbreak is no surprise, since most cholera outbreaks today are caused by this
biotype.

The Ogawa serotype allows characterization of the antigenic profile of the implicated strain. The
other serotypes include Inaba and Hikojima. These do not have a major importance in practice.

Part 11. Drawing conclusions and formulating recommendations

The cohort study is now complete; results indicate that the risk of cholera is significantly higher in
persons using the municipal water system as a primary source of drinking water (Table 3) than
those not using it as a primary source of drinking water. Thus the analytical epidemiological data
support the hypothesis that the municipal water supply caused the outbreak.

Table 2: Risk of cholera according to exposure to the Municipal Water system, Kanchrapara
Municipality, North 24 Parganas District, West Bengal, India, 2004
Developed Did not Total
cholera develop
cholera
Use the municipal water system as 189 742 931
a primary source of drinking water
Do not use the municipal water 5 365 370
system as a primary source of
drinking water
Total 194 1207 1301

(Relative risk: 15; 95% Confidence Interval 6.2-36)

Question 11a
How can the epidemiological, environmental and laboratory investigations be integrated to draw
conclusions for the seventh step of the investigation?

Question 11b
What short-term and long-term recommendations can be formulated?

Cholera India, May 2007 17

Discussion for Part 11

Once the results of the investigation came together to explain the outbreak, the epidemiologist
organized a debriefing that was attended by hospital clinicians, city water engineers, the district
authorities and the chief medical officer. The laboratory was invited to the debriefing, but couldn’t
attend, so he also provided a debrief over the phone to the lab. During the debrief, he presented
the evidence and his interpretation and rationale for drawing his conclusions, and proposed
appropriate interventions and recommendations.

The epidemiological and laboratory data support the hypothesis that contamination of the
municipal water system supplying the Kulia road and Dharmanabar colony areas was the source of
the outbreak. The environmental assessment suggested that the contamination was secondary to
a leak that sucked sewage-contaminated water into the municipal water supply. The sewage
contained Vibrio cholerae. Since the water was un-chlorinated, it led to the cholera outbreak.
Identifying the leak on 12 October followed by prompt repair, including heavy chlorination, ended
the outbreak, with the number of new cases decreasing rapidly after the intervention.

On the basis of these conclusions, the epidemiologist formulated a number of recommendations,

including:

Short term
 Investigate and promptly repair all leaks reported in the municipal water supply.
 Use this outbreak as an example to other municipalities of the effectiveness of rapid
intervention to contain and control outbreaks.

Long term
 Ensure routine chlorination of the municipal and railway water supply. Chlorination should
reach 0.5 mg / litre for all sampling points in a piped water system, 1 mg / litre at all standposts
for systems with standposts and 2mg/ litre at filling if the water is distributed with tanker trucks.
Error: Reference source not found
 Discuss with municipal authorities what could be done to improve the sanitation in the area. 8
 Continue with the promotion of other general hygiene measures for the prevention of diarrheal
diseases, including hand washing, and possibility, safe water systems. 9

Epilogue
Before leaving the field, the epidemiologist wrote a short report summarizing his findings, his
conclusions and recommendations. He thanked all outbreak team members for their collaboration.
He left this report with all the stakeholders. He also mailed a copy of that report to the laboratory
that participated in the investigation.

Cholera India, May 2007 18

References

Cholera India, May 2007 19

1
WHO. Guidelines for cholera control. WHO Geneva, Switzerland, 1993.
2
WHO. WHO recommended surveillance standards. Second edition, October 1999. WHO Geneva,
Switzerland, WHO/CDS/CSR/ISR/99.2.
3
WHO. Guidelines for the collection of clinical specimens during field investigation of outbreaks. WHO
Geneva, Switzerland, WHO/CDS/CSR/EDC/2000.4.
4
WHO. Laboratory biosafety manual. Third edition. WHO Geneva, Switzerland,
WHO/CDS/CSR/LYO/2004.11.
5
WHO. Guidelines for the collection of clinical specimens during field investigation of outbreaks. WHO
Geneva, Switzerland, WHO/CDS/CSR/EDC/2000.4.
6
World Health Organization – Centers for Disease Control and prevention (CDC): Laboratory Methods
for the Diagnosis of Epidemic Dysentery and Cholera. 

7
World Health Organization. Guidelines for drinking water quality Vol. 1 Recommendations, second
edition. World Health Organization 1993.
8
Francys R, Pickford J, Reed R. A guide to the development of on-site sanitation. Geneva, World
Health Organization, 1992.
9
Luby S, Agboatwalla M, Raza A, Sobel J, Mintz E, Baier K, Rahbar M, Qureshi S, Hassan R, Ghouri
F, Hoekstra RM, Gangarosa E.: A low-cost pilot intervention for cleaner drinking water in Karachi,
Pakistan. Int J Infect Dis 2001; 5:144-50.