**Botulism in Argentina**

STUDENT'S VERSION

+-----------------------------------------------------------------------+
| **Original investigators:** Rodrigo G. Villar, MD,^1^ Roger L.        |
| Shapiro, MD,^1^ Silvina Busto, MD, MPH,^2^ Clara Rive-Posse, MD,      |
| MPH,^3^ Guadalupe Verdejo MD, MPH,^4^ Maria Isabel Farace, DVM,^5^    |
| Francisco Rosetti, MS,^5^ Jorge A. San Juan, MD,^6^ Carlos Maria      |
| Julia, MD,MPH,^3^ John Becher, RPh,^1^ Susan E. Maslanka, PhD,^1^     |
| David Swerdlow, MD^1^                                                 |
|                                                                       |
| ^1^Centers for Disease Control and Prevention, ^2^Ministero de        |
| Salud-Region V, ^3^Ministero de Salud y Accion Social, ^4^Pan         |
| American Health Organization, ^5^Departamento de Bacteriologia,       |
| Instituto Nacional de Enfermedades Infecciosas, ^6^Hospital F. J.     |
| Muñiz, Buenos Aires, Argentina                                        |
|                                                                       |
| **Case study and instructor's guide created by:** Jeanette K.         |
| Stehr-Green, MD                                                       |
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> *NOTE: This case study is based on a real-life outbreak investigation
> undertaken in Buenos Aires, Argentina, in 1998. Some aspects of the
> original outbreak and investigation have been altered, however, to
> assist in meeting the desired teaching objectives and allow completion
> of the case study in less than 3 hours.*
>
> *Students should be aware that this case study describes and promotes
> one particular approach to foodborne disease outbreak investigation.
> Procedures and policies in outbreak investigations, however, can vary
> from country to country, state to state, and outbreak to outbreak.*
>
> It is anticipated that the epidemiologist investigating a foodborne
> disease outbreak will work within the framework of an "investigation
> team" which includes persons with expertise in epidemiology,
> microbiology, sanitation, food science, and environmental health. It
> is through the collaborative efforts of this team, with each member
> playing a critical role, that outbreak investigations are successfully
> completed.
>
> *We invite you to send us your comments about the case study by
> visiting our website at
> [http://www.phppo.cdc.gov/phtn/casestudies]{.underline}. Please
> include the name of the case study with your comments.*

**April 2002**

**U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES**

**Public Health Service**

**Centers for Disease Control and Prevention**

**Atlanta, Georgia 30333**

**STUDENT'S VERSION**

**Botulism in Argentina**

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| **Learning objectives:**                                              |
|                                                                       |
| After completing this case study, the student should be able to:      |
|                                                                       |
| 1.  describe outbreak situations in which acute control measures      |
|     should be undertaken                                              |
|                                                                       |
| 2.  communicate information on an outbreak or outbreak investigation  |
|     and write a press release                                         |
|                                                                       |
| 3.  given the leading hypothesis(es) in an outbreak, develop a        |
|     questionnaire                                                     |
|                                                                       |
| 4.  given details on the origin, distribution, and preparation of an  |
|     implicated food item, identify critical points for the control of |
|     contamination and microbial survival and growth                   |
|                                                                       |
| 5.  discuss possible barriers to implementation of specific           |
|     interventions following an outbreak investigation                 |
|                                                                       |
| 6.  describe measures that can be used to monitor the success of an   |
|     intervention                                                      |
|                                                                       |
| 7.  describe the occurrence, signs and symptoms, and control of       |
|     foodborne botulism                                                |
|                                                                       |
| ```{=html}                                                            |
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| ```                                                                   |
| 1.                                                                    |
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**PART I - OUTBREAK DETECTION**

Foodborne botulism is a severe illness that results from the ingestion
of a preformed toxin produced by a bacterium, *Clostridium botulinum*,
in contaminated food. Death can occur in up to 60% of untreated cases;
supportive care and prompt administration of antitoxin have reduced
mortality in the United States to less than 10%. Outbreaks of botulism
have been linked to improperly preserved vegetables, fruits, and meats
including fermented fish products, sausages, smoked meat, and seafood.

On January 13, 1998, an infectious diseases physician at a Buenos Aires
hospital telephoned the Directorate of Epidemiology of the Argentine
Ministry of Health (MOH) to report two possible cases of botulism. The
patients, both men, presented with drooping eyelids, double vision,
difficulty swallowing, and respiratory problems. One patient had onset
of symptoms on January 5 and the other on January 6. The physician had
drawn sera and collected stool specimens from the men to test for
botulinum toxin but no results were available.

[Question 1:]{.underline} As a public health practitioner in Argentina,
what are the major concerns raised by these two possible cases of
botulism in Buenos Aires?

The clinical syndrome of botulism is dominated by neurologic signs and
symptoms. Dryness of the mouth, drooping eyelids, and blurred and double
vision are usually the earliest neurologic complaints. These initial
symptoms may be followed by disturbances in speech, difficulties
swallowing, and peripheral muscle weakness. If respiratory muscles are
involved, ventilatory failure and death may result unless supportive
care is provided. The average incubation period for botulism is 18-36
hours, but symptoms can occur as early as six hours or as late as 10
days after exposure.

Because botulism is rare, many physicians are unfamiliar with its
presentation. As a result, patients with botulism can be misdiagnosed as
other illnesses (e.g., stroke, myasthenia gravis, Guillain‑Barré
syndrome) delaying the administration of life-saving botulinum antitoxin
for days and increasing the mortality rate among cases.

[Question 2:]{.underline} How might you go about swiftly determining if
there are other cases of botulism associated with the cases in Buenos
Aires?

The Directors of the National Laboratory and the Environmental Health
and Sanitation Program were notified of the possible cases of botulism.
The two patients, still in the hospital, were interviewed by an MOH
epidemiologist.

Upon questioning, it was learned that both patients were drivers for the
same bus company and drove the same route and shift. The patients knew
each other but worked on different days of the week. They had not eaten
together in more than a month.

To find additional cases, the MOH contacted all employees of the bus
company with the ill drivers to see if any had symptoms suggestive of
botulism. Hospitals in the area of Buenos Aires, where the two cases
occurred, were asked to report any patients with acute neurologic
illnesses that could be botulism. Family members of cases were
questioned about whether they also had symptoms of botulism.
Additionally, the MOH developed a press release for distribution to the
local news media.

[Question 3A:]{.underline} What key points would you include in the
press release?

[Question 3B:]{.underline} Who should be involved in developing the
press release or notified before its distribution?

On January 14, the MOH distributed the following press release:[^1]

*On Monday, January 13, two bus drivers, Pablo Esteban and Juan Rojas,
from south central Buenos Aires were diagnosed with botulism by an
infectious diseases physician at Hospital F. J. Muñiz. The men had been
ill for several days before the diagnosis was made. The attending
physician, Dr. Jorge San Juan reported that botulinum antitoxin was
requested from the U. S. Centers for Disease Control and Prevention last
night.*

> *Because botulism is potentially fatal if untreated, the Argentine
> Ministry of Health is working with local public health officials and
> health care providers to identify the source of the botulism.
> Officials worry that other persons may have been exposed and need
> treatment or that the source may still exist and cause more people to
> become ill.*
>
> *Botulism is a rare but serious paralytic illness caused by a nerve
> toxin produced by a bacterium, [Clostridium]{.underline}
> [botulinum]{.underline}. The symptoms of botulism can easily be
> confused with other illnesses and include diplopia, ptosis, dysphagia,
> dysarthria, and muscle weakness. If untreated, these symptoms may
> progress to cause paralysis of the arms, legs, trunk and respiratory
> muscles, and ultimately death. If diagnosed early, botulism can be
> treated with an antitoxin which blocks the action of the toxin
> circulating in the blood.*
>
> *Previous outbreaks of botulism in Argentina have been caused
> primarily through eating improperly preserved vegetables and meats.
> Implicated foods include ham, red and green peppers, vigcacha (an
> Andean rodent), eggplant, cucumbers, palm hearts, tomatoes, peaches,
> spinach, and a type of cheese with onions. It is not yet known what
> specific food may have caused the botulism in this outbreak but both
> men are bus drivers for the same company, Arriba Bus, Inc., and drive
> the same route and shift.*
>
> *Persons who feel they or their relatives might have symptoms of
> botulism are asked to contact the Directorate of Epidemiology of the
> Argentine Ministry of Health at xxxx-xxxx.*

[Question 4:]{.underline} Critique the press release. How might the
press release impact the outbreak investigation?

Seven additional patients with neurologic signs consistent with botulism
were identified. Five of the patients had sought medical attention and
four were hospitalized. Working diagnoses for these patients at the time
the initial two cases were discovered included myasthenia gravis (1),
Guillain-Barré syndrome (2), stroke (1), and diabetic complications (1).

Botulinum toxin was identified in sera and/or stool from three patients,
including one of the original cases reported on January 13.

All patients were drivers from the same bus company as the original
cases and drove the same route. From initial reports, all had eaten at a
home located at the terminal stop of the bus route where the drivers
stopped during their breaks. Approximately 58 bus drivers worked this
route; 27 in the morning shift, 16 in the afternoon shift, and 15 in the
evening shift.

[Question 5:]{.underline} Would you initiate any control measures at
this time? What criteria would you consider in implementing control
measures so early in an investigation?

**PART II - DESCRIPTIVE EPIDEMIOLOGY AND HYPOTHESIS GENERATION**

Staff from the local health department where the terminal stop of the
bus route was located were invited to participate in the investigation.

Physicians attending the cases of botulism were asked to provide
demographic and clinical information on their patients. (Table 1)

Table 1. Characteristics of cases of botulism, Buenos Aires, January
1998.

  --------- --------- -------- --------- ------------- -------------------------
  Patient   Age       Gender   Work      Onset of      Symptoms
  No.       (years)            shift     neuro-logic   
                                         symptoms      

  1         42        M\*      Morning   January 6     blurred vision, double
                                                       vision, drooping eyelids,
                                                       upper and lower extremity
                                                       weakness, respiratory
                                                       difficulty, fatigue

  2         31        M        Morning   January 5     blurred vision, double
                                                       vision, drooping eyelids,
                                                       upper and lower extremity
                                                       weakness

  3         23        M        Morning   January 9     blurred vision, drooping
                                                       eyelids, upper extremity
                                                       weakness, fatigue

  4         46        M        Morning   January 8     drooping eyelids,
                                                       difficulty speaking

  5         54        M        Morning   January 5     blurred vision, double
                                                       vision, drooping eyelids,
                                                       difficulty speaking,
                                                       respiratory difficulty

  6         49        M        Morning   January 10    blurred vision, drooping
                                                       eyelids, difficulty
                                                       speaking

  7         31        M        Morning   January 15    blurred vision, double
                                                       vision, drooping eyelids,
                                                       upper and lower extremity
                                                       weakness, respiratory
                                                       difficulty, fatigue

  8         44        M        Morning   January 14    respiratory difficulty,
                                                       fatigue, drooping
                                                       eyelids,

  9         24        M        Morning   January 12    drooping eyelids, fatigue
  --------- --------- -------- --------- ------------- -------------------------

\*M=male gender

[Question 6A:]{.underline} Summarize the demographic and clinical
information on these patients and draw

an epidemic curve.

[Question 6B:]{.underline} When was the most likely period of exposure
among these cases?

In hypothesis-generating interviews with cases and other bus drivers,
being a driver on the morning shift of the bus route and eating at the
terminal home of the route were the only common exposures among cases.
No cases of botulism occurred among bus drivers from the afternoon or
evening shift of the route. Bus drivers from those shifts did not
usually eat at the terminal home because it was only open for lunch.

The investigators hypothesized that the outbreak was limited to morning
shift bus drivers and resulted from eating or drinking something at the
home at the terminal stop of the particular bus route between January 3
and 7.

[Question 7:]{.underline} What type(s) of study would you use to
investigate this hypothesis? Why?

**PART III - DESIGNING AN EPIDEMIOLOGIC STUDY TO TEST THE HYPOTHESIS**

To identify the source of the outbreak, investigators undertook a
retrospective cohort study among bus drivers who drove the morning shift
of the bus route. Data were collected from January 15-19.

Investigators defined a confirmed case of botulism as a bus driver from
the morning shift of the bus route with a serum or stool sample that
demonstrated botulinum toxin or yielded *Clostridium botulinum* with
onset of symptoms between January 5 and 15. A probable case was defined
as acute cranial nerve dysfunction (e.g., blurred vision, double vision,
drooping eyelids, problems swallowing) with no laboratory confirmation
in this group of drivers during the same period. The comparison group
consisted of all bus drivers from the morning shift of the implicated
bus route who had no acute neurologic symptoms suggestive of botulism.

After consultation with the local health department where the terminal
stop of the bus route was located and the bus company management,
investigators developed a structured questionnaire for the epidemiologic
study.

[Question 8:]{.underline} What general types of information would you
include in the questionnaire?

[Question 9:]{.underline} What steps would you take to develop the
questionnaire for this investigation.

[Question 10:]{.underline} Using information on foods served at the
terminal stop home from January 3-7 (Appendix 1), draft questions for
food exposure for this study.

Investigators conducted interviews with each of the drivers of the
morning shift of the bus route to complete the questionnaires.

[Question 11:]{.underline} If the investigators had decided on
self-administration of the questionnaire, what changes might need to be
made to it?

**PART IV - ANALYSIS AND INTERPRETATION OF EPIDEMIOLOGIC RESULTS**

The following food exposure information was collected through the cohort
study. On January 19, the information was tabulated by epidemiologists
from the Argentine MOH. (Table 2)

Table 2. Foods eaten by ill and well bus drivers at the home at the
terminal bus stop, January 3-7, 1998. (N=21)

  ------------------------- --------- ------------ --------- -------------
  Food item                 Ate item               Did not   
                                                   eat item  

                            Ill       Well         Ill       Well

  Bologna                   1         0            8         12

  Hot dog                   1         1            8         11

  Matambre\*                9         2            0         10

  Mate\*\*                  4         4            5         3

  Processed Ham             2         3            7         9

  Sauce                     7         2            2         10

  Salami                    1         1            8         11

  Solid ham                 2         3            7         9
  ------------------------- --------- ------------ --------- -------------

\*Matambre is a traditional meat roll in Argentina.

\*\*Mate is green tea.

[Question 12:]{.underline} Calculate the appropriate measures of
association for these exposures.

[Question 13:]{.underline} Interpret the results. What further data
analysis/information might help?

**PART V - ENVIRONMENTAL STUDIES AND FOOD INVESTIGATION**

Matambre is a traditional Argentine dish prepared from meat, vegetables,
spices, and eggs. In a traceback of the implicated matambre, the MOH
discovered that it originated from a small scale producer located not
far from the terminal stop of the bus route. The matambre was purchased
at a local market on January 3 by the owners of the home that served the
matambre. The MOH initiated an environmental health assessment of the
matambre to identify production factors that could have contributed to
the occurrence of botulism.

A complete environmental health assessment is not a cursory inspection
of operations and sanitary conditions as is performed for the licensing
of a food establishment, but focuses on the suspect food or meal and
follows it from its raw ingredients to consumption by the customer. The
objective of the environmental health assessment is to identify critical
points where the implicated food could have become contaminated or
microbial survival and growth in the food could have occurred, determine
why these conditions existed, and identify appropriate interventions.
The factors in Table 3, often found in these assessments, have been
associated with an increased risk of foodborne disease.

Table 3. Factors that commonly contribute to outbreaks of foodborne
diseases, from Bryan et al., 1987.

+-----------------------+-----------------------+-----------------------+
| Contamination         | Survival              | Growth                |
+-----------------------+-----------------------+-----------------------+
| raw foods that are    | inadequate cooking    | inadequate            |
| contaminated          |                       | refrigeration         |
|                       | inadequate reheating  |                       |
| infected foodhandler  |                       | improper cooling      |
|                       | inadequate            |                       |
| unclean equipment     | acidification         | inadequate            |
|                       |                       | hot-holding           |
| cross-contamination   |                       |                       |
|                       |                       | preparation too far   |
| contaminated foods    |                       | in advance            |
| eaten                 |                       |                       |
|                       |                       | of serving            |
| raw or lightly cooked |                       |                       |
|                       |                       | use of leftover foods |
| inappropriate         |                       |                       |
| container for food    |                       | inadequate            |
|                       |                       | acidification         |
| unsafe sources        |                       |                       |
|                       |                       | high water content    |
| added poisonous       |                       |                       |
| chemicals             |                       | inadequate curing     |
|                       |                       | salt                  |
| natural toxicant      |                       |                       |
|                       |                       | environment that      |
| poor dry-storage      |                       | provides              |
| practices             |                       |                       |
|                       |                       | favorable conditions  |
|                       |                       | for                   |
|                       |                       |                       |
|                       |                       | pathogen (e.g.,       |
|                       |                       | anaerobic             |
|                       |                       |                       |
|                       |                       | packaging)            |
+-----------------------+-----------------------+-----------------------+

[Question 14:]{.underline} What types of activities do you think you
would undertake as part of an environmental health assessment on the
matambre? What equipment would you want to have?

*Clostridium botulinum* is a spore-forming obligate anaerobic bacterium
(i.e., it cannot grow in the presence of oxygen). The spores are
widespread in soil and dust worldwide. The toxin is produced in
improperly canned, low-acid or alkaline foods and in pasteurized and
lightly cured foods held without adequate refrigeration, especially in
airtight containers. The toxin is destroyed by boiling; inactivation of
spores requires much higher temperatures.

Food safety inspectors from the local health department initiated the
environmental health assessment on January 20. The home at the terminal
stop of the bus route was not formally licensed or equipped as a
restaurant. Perishable foods, such as the matambre, were kept in two
large refrigerators inside the home. Although the refrigerators were set
at the coldest possible setting, temperatures measured inside the
refrigerators were 9C and 10C (48F and 50F, respectively).

The home owner reported the most recent matambre served in the home
weighed approximately 4 kg. The matambre was cut into about 15 slices
and was served in sandwiches, usually with a spicy sauce; no other
condiment or ingredient was added to the sandwiches. No matambre was
available for testing.

The implicated matambre was bought at a local market where it had been
stored in a refrigerator. The market had no temperature records or sales
receipts; however, some customers reported that the matambre had
recently been sold at reduced prices because of power outages.

The market purchased matambre from a small scale commercial producer who
made matambre and processed hams in his home. To make matambre, the
producer placed a slab of raw beef (1- to 3-cm thick) on a stainless
steel table. Ingredients included raw sliced carrots, hard-boiled eggs,
salt, red pepper flakes, dried oregano, and commercial potato flour. The
meat was rolled up around the vegetables and eggs to make an
approximately 10 x 30 cm cylinder. The meat roll was placed into a
rectangular stainless steel pan to keep ingredients inside during
cooking. Between 10 and 15 matambre in individual steel pans were
immersed together in water heated to 70 to 80C (158 to 176F) and
cooked approximately 4 hours. The matambre was never brought to a boil.
After cooking, the water was drained and the temperature was checked to
ensure an internal temperature of about 68C (154F). The producer
placed each warm matambre in plastic wrap, squeezed out the air, and
sealed the plastic with heat. The plastic wrapped matambre were allowed
to cool, placed in a walk-in refrigerator, and were stored for up to 2
weeks before being sold to either supermarkets or directly to consumers.

[Question 15:]{.underline} Identify the foodhandling practices for the
matambre which were most likely to contribute to the development of
botulism.

The producer reported making matambre every 2 weeks in batches of 15-20
each time. The last batch produced before the outbreak was made in early
December. No matambre were available from that batch.

[Question 16:]{.underline} What control measures would you initiate at
this time? What difficulties might you encounter?

**PART VI - CONTROL**

After inspection by local food safety officials, the facility producing
the matambre was closed. The producer was unable to provide receipts or
a distribution list with locations where his products were sold. He
reported that most of his clients distributed his products in the
western greater Buenos Aires area. The producer's matambre was not
labeled in any way to indicate the source or date of production, so a
recall of any remaining matambre was not deemed feasible.

Based on data maintained by the MOH, botulism is not an uncommon
occurrence in Argentina. During the years 1979-97, 277 cases of botulism
were reported; for most, the source of the exposure was undetermined. In
1997, 23 patients with suspected botulism were reported (of which 13
\[57%\] died); about the same number of laboratory-confirmed cases were
identified in the United States, which has 10 times the population of
Argentina.

[Question 17:]{.underline} Given the scope of the problem, what longer
term interventions might help decrease the number of botulism cases in
Argentina?

Because of the relatively high incidence and case fatality ratio for
botulism in Argentina, the MOH and Centers for Disease Control and
Prevention (CDC) collaborated to establish a botulism surveillance and
antitoxin release system in Argentina. The system components
included: 1) the establishment of a local stock of antitoxin, 2) a
mechanism for antitoxin distribution within the country, 3) emergency
notification and response for suspect cases (including urgent
epidemiologic investigation of the possible food source), and 4)
laboratory confirmation of suspect cases.

[Question 18:]{.underline} What is the rationale behind the components
of this control effort?

[Question 19:]{.underline} How might you monitor the Argentine botulism
surveillance and antitoxin release system over time to determine if it
is effective?

**EPILOGUE**

Although consumption of matambre is an established tradition in
Argentina, it is usually consumed fresh and is not generally intended
for pickling or long-term preservation. Matambre produced by licensed,
commercial facilities use nitrites, acidifiers, or other preservatives
to prevent bacterial growth; the implicated matambre lacked these.
Insufficient cooking, vacuum packing in heat-shrinked wrap, and
inadequate refrigeration may have provided conditions for live spores to
germinate and produce toxin.

The matambre that is believed to be the cause of the outbreak was cooked
at relatively low temperatures (78-80C or 158-176F ) over a time
period of approximately 240 minutes, too short to kill all *C.
botulinum* spores. These spores are difficult to destroy using
conventional cooking techniques. In fact, nonkilling heat shock and the
lack of preservatives or acidifiers may even enhance germination and
toxin elaboration.

To safely prepare foods for canning or long-term storage, the U.S.
Department of Agriculture recommends that all low-acid foods (i.e.,
foods with a pH \> 4.6, including meats, seafood, poultry, milk, and
fresh vegetables) be sterilized at temperatures of 116 to 121C
(240-250F) in pressure canners operated at 0.66 to 0.97 atm (10-15
lb/in^2^). At these temperatures, the time needed to destroy bacteria in
low-acid canned food ranges from 20-100 minutes. The exact time depends
on the kind of food being canned, the way it is packed, and the amount
of food being cooked.

**REFERENCES**

Villar RG, Shaprio RL, Busto S, et al. Outbreak of Type A botulism among
bus drivers and development of a botulism surveillance and antitoxin
release system in Argentina. JAMA 1999; 281:1334-1340.

Bryan FL, Anderson HW, Cook OD, et al. Procedures to investigate
foodborne illness. International Association of Milk, Food, and
Environmental Sanitarians, Inc.: Ames Iowa; 1987.

Centers for Disease Control and Prevention: Botulism in the United
States, 1899-1996. Handbook for Epidemiologists, Clinicians, and
Laboratory Workers, Atlanta, GA. Centers for Disease Control and
Prevention, 1998.

Shapiro RL, Hatheway C, Becher J, Swerdlow DL. Botulism surveillance and
emergency response. A Public Health Strategy for a Global Challenge.
JAMA 1997; 278:433-435.

Shapiro RL, Hatheway C, Swerdlow DL. Botulism in the United States: A
clinical and epidemiologic review. Annals of Internal Medicine 1998;
129:221-228.

St. Louis ME, Peck SHS, Bowering D, et al. Botulism from chopped garlic:
Delayed recognition of a major outbreak. Annals of Internal Medicine
1988; 108:363-68.

Townes JM. Cieslak PR, Hatheway CL, et a;. An outbreak of Type A
botulism associated with a commercial cheese sauce. Annals of Internal
Medicine 1996;125:558-63.

**APPENDIX 1**

Foods served to bus drivers in home at terminal stop of bus route in the
first week of

January:

Bologna

Hot dogs

Matambre\*

Mate\*\*

Processed ham

Sauce

Salami

Solid ham

\*Matambre is a traditional meat roll in Argentina. At the terminal stop
it was sliced and served in sandwiches, usually with a spicy sauce.

\*\*Mate is green tea.

[^1]: ^\*^ This is not the press release distributed by the Ministry of
    Health but was developed specifically for this exercise.