Case Study 4: Influenza A, H2N2 Outbreak in a Laboratory
Facilitator Guide
SITUATION
In the late 1950s, the H2N2 strain of the influenza A virus was responsible for the deaths
of between one and four million people worldwide during what is commonly referred to
as the Asian influenza pandemic. H2N2 continued to circulate in humans until 1968,
when it disappeared with the emergence of influenza A/H3N2, which sparked the Hong
Kong influenza pandemic. In present day vaccinations, the H2N2 virus is not included as
part of the FDA-approved trivalent influenza vaccine. As a result, individuals born post1968 exhibit little to no immunity against influenza A/H2N2 strains.
Proficiency testing (PT) is used to test a laboratory’s competency in performing a
particular diagnostic test. Laboratories participating in a PT exercise receive samples
containing specified material with an identity and quantity known only to those
orchestrating the PT exercise. The laboratory analyzes the samples according to a predetermined protocol or standard operating procedure and reports the results to the
exercise organizers. The organizers then inform the participating laboratory how closely
the results reported match the accepted values. If and when necessary, the laboratory can
take appropriate actions to improve its performance and capabilities.
UPDATE 1
You are the director of the State Public Health Laboratory. On April 8, 2005, the College
of American Pathologists (CAP) sends a fax to various national and international public
health laboratories, including your State Public Health Laboratory, regarding a recently
disseminated PT exercise.
The fax alerts you that one of the specimens included in this exercise has been identified
by a local laboratory in Canada as influenza A subtype H2N2. Approximately 4,600 PT
samples containing H2N2 have been disseminated to laboratories between October 2004
and March 2005. Your laboratory received the PT samples one week ago. Some of your
laboratorians, unaware that the samples contain the dangerous H2N2 virus, have already
begun performing analysis on the PT specimens.
1. How worrisome do you consider this error to be?
Suggested answer: Thanks to the routine safety precautions inside the laboratory, the
risk of H2N2 spreading outside the laboratory is probably quite small. However, the fact
that individuals born after 1968 have little to no immunity to this particular strain is
worrisome. If H2N2 were contracted by a laboratory worker and spread outside the
laboratory setting, the potential could exist for an outbreak—or even a pandemic!
2. What modes of transmission are associated with influenza viruses?
Suggested answer: Influenza is primarily transmitted from person to person via large
virus-laden droplets generated when an infected person coughs, sneezes, or talks. These
droplets can settle on the mucosal surfaces of the conjunctivae and/or upper respiratory
tracts of susceptible persons who are close to (i.e., within 3 feet) infected persons.
Transmission can also occur through direct contact or indirect contact with respiratory
secretions, such as touching surfaces contaminated with influenza virus and then
touching the eyes, nose, or mouth.
Adults may be infectious and able to spread influenza to others from the day before
getting symptoms to approximately 5 days after symptoms start, while young children
may be infectious for 10 or more days after onset of symptoms.
3. What laboratory hazards might lead to the accidental transmission of an influenza
virus from a specimen to a laboratory worker?
Suggested answer: The primary laboratory hazard involves the inhalation of flu virus
from aerosols generated by infecting animals or by aspirating, dispensing, mixing,
centrifuging, or otherwise manipulating samples infected with the influenza virus. In
addition, laboratory infection can result from direct inoculation of mucus membranes
through virus contaminated gloves following the handling of flu-containing specimens.
4. What are laboratory biosafety levels? What do you know about each one?
Suggested answer: Four distinct biosafety levels have been established to achieve
“containment,” or safe methods for managing infectious materials in the laboratory
environment where they are being handled or maintained. Containment acts to reduce or
eliminate the possibility of exposure of laboratory workers, other persons, and the
outside environment to potentially hazardous agents. Therefore, the three elements of
containment include laboratory practice and technique, safety equipment, and facility
design.
The biosafety levels consist of combinations of the three elements of containment
depending upon the agent being analyzed, appropriate methods for the operations
performed, the documented or suspected routes of transmission, and the laboratory
function or activity. The recommended biosafety level(s) for an organism represents
those conditions under which the agent ordinarily can be safely handled. See Table 1
below for a summary of the biosafety levels.
The laboratory director is specifically and primarily responsible for assessing the risks
and appropriately applying the recommended biosafety levels.
Table 1. Summary of Recommended Biosafety Levels for Infections Agents
BSL
Agents
1
Not known to
consistently cause
diseases in healthy
adults
 Agents associated
with human disease
 Routes of
transmission include
percutaneous injury,
ingestion, mucous
membrane exposure
Standard
microbiological
practices
 Indigenous or
exotic agents with
potential for aerosol
transmission
 Disease may have
serious or lethal
consequences
BSL-2 plus:
 Controlled
access
 Decontamination
of all waste
 Decontamination
of laboratory
clothing before
laundering
 Baseline serum
2
3
Practices
BSL-1 plus:
 Limited access
 Biohazard
warning signs
 Sharps
precautions
 Biosafety
manual defining
any needed
decontamination of
waste or medical
surveillance
policies
Primary Barriers &
Safety Equipment
None required
Facilities
(Secondary Barriers)
Open bench and sink
required
Primary barriers:
 Class I or II biological
safety cabinets (BSCs)
or other physical
containment devices
used for all
manipulations of agents
that cause splashes or
aerosols of infectious
materials
Personal protective
equipment (PPEs):
 Laboratory coats;
gloves; face protection
as needed
Primary barriers:
 Class I or II BSCs or
other physical
containment devices
used for all open
manipulation of agents
PPEs:
 Protective laboratory
clothing; gloves;
respiratory protection as
needed
Primary barriers:
 All procedures
conducted in Class III
BSCs or Class I or II
BSCs in combination
with full-body, airsupplied, positive
pressure personnel
suit
BSL-1 plus:
 Autoclave available
BSL-2 plus:
 Physical separation
from access corridors
 Self-closing,
double-door access
 Exhaust air not
recirculated
 Negative airflow
into laboratory
 Dangerous/exotic
BSL-3 plus:
BSL-3 plus:
agents which pose
 Clothing change
 Separate building or
high risk of lifebefore entering
isolated zone
threatening disease
 Shower on exit
 Dedicated supply
 Aerosol All material
and exhaust, vacuum,
transmitted
decontaminated on
and decontamination
laboratory infections exit from facility
systems
have occurred; or
 Other requirements
related agents with
outlined in the text
unknown risk of
transmission
From Biosafety in Microbiological and Biomedical Laboratories (BMBL) 5th Edition, available at:
http://www.cdc.gov/od/ohs/biosfty/bmbl5/bmbl5toc.htm.
4
5. What laboratory biosafety level would be appropriate for conducting routine
influenza testing?
Suggested answer: Routine influenza testing in the laboratory should adhere to Biosafety
Level 2 (BSL-2) guidelines, as recommended by the Biosafety in Microbiological and
Biomedical Laboratories (BMBL) 5th Edition. BSL-2 is suitable for work involving
agents that pose moderate hazards to personnel and the environment. BSL-2 guidelines
require that: 1) laboratory personnel have specific training in handling pathogenic
agents and are supervised by scientists competent in handling infectious agents and
associated procedures; 2) access to the laboratory is restricted when work is being
conducted; and 3) all procedures in which infectious aerosols or splashes may be created
are conducted within biological safety cabinets or other physical containment equipment.
6. Group Brainstorm: Have one member of the group write ideas on the flip chart.
What pieces of personal protective equipment (PPE) should be used when conducting
routine influenza testing in the public health laboratory setting?
Suggested answer: Possible answers include the following.
 Class II biological safety cabinet
 Fume hood
 Glove box
 Protective laboratory gowns, coats, smocks, or uniforms
 Eye and face protection, such as goggles, masks, face shields, or splatter guards
 Gloves
 Respiratory equipment, such as powered air-purifying respirators (PAPR), selfcontained breathing apparatuses (SCBA), N95 respirators, etc.
UPDATE 2
After reading the fax, you make your way to the microbiology/virology section of the
laboratory where the PT exercise was being handled. You share the news with staff
members there, and discuss how to proceed with the situation (i.e., safely dispose of the
dangerous samples).
7. Who are the stakeholders? Is there a need to communicate with outside stakeholders
in this situation? If so, which stakeholders?
Suggested answer: It would be appropriate to engage outside stakeholders at this point,
even if only to make them aware of the situation. Initial stakeholders could include CAP
(the organization administering the PT exercise), your local and state health
departments, and the CDC. Since the H2N2 samples are exceedingly dangerous and you
have not yet determined how to dispose of them, it would be worthwhile to notify law
enforcement, as the samples could pose a security threat if they fell into the wrong hands.
8. Which, if any, laboratory staff members could have been exposed to the H2N2 virus?
Suggested answer: Those possibly exposed to the virus could include anyone working in
the area of the microbiology/virology section during the time that the H2N2 samples
were being analyzed. Even though the laboratorians should have been wearing PPE
while working with the H2N2 samples, they should still be considered at risk. Since they
were unaware that they were working with H2N2 when they began testing the samples,
they may not have been wearing all of the necessary PPE or working in appropriate
biosafety level conditions. Even if the laboratorians were donning appropriate PPE,
there is always a chance that the PPE was not used correctly or consistently.
Additionally, those working in the general vicinity of the area where H2N2 samples were
being tested may not have been wearing PPE at all times, as they may have been
performing routine tests that did not require them to wear PPE beyond gloves or safety
glasses. Close contacts and family members of all these individuals are also at risk for
secondary exposure.
9. What surveillance strategies will you use to identify people who have been exposed
or infected with H2N2?
Suggested answer: It is essential that surveillance be implemented within the laboratory
to monitor any laboratory staff presenting with influenza-like illness. Such a system
could closely track absenteeism within the laboratory and involve conducting telephone
calls to absent staff members to monitor symptoms. When you make plans regarding how
long staff members will be monitored for signs and symptoms of flu, keep in mind that the
incubation period for flu is one to three days. It would also be worthwhile to inquire as
to the overall health of family members and other close contacts of potentially exposed
employees to determine if the infection could be spreading. As an extra precaution,
surveillance measures might also involve testing all laboratory staff potentially exposed
to the H2N2 virus to determine if exposure actually occurred.
10. Is there a potential that anyone could have been exposed to the H2N2 virus prior to
the H2N2 samples arriving to the laboratory? If so, how could this have happened?
Suggested answer: If the specimens were not properly packaged and shipped, there is a
risk that the laboratory courier or staff in the sample processing and receiving area of
the lab may have been exposed. For example, if the specimens were transported in
incorrect or improperly sealed containers, they may have posed a risk to those handling
their delivery. It will be important to speak with the courier service and any laboratory
staff involved in sample accession to ensure that the packages showed no signs of leakage
or seepage, were packaged in compliance with mail and commercial regulations for
transport of infectious material, etc. Depending upon this information, it may be
necessary to follow up with additional potentially-exposed individuals.
11. How would a chain of custody help you easily determine who had contact with the
samples until the shipment reached the laboratory?
Suggested answer: “Chain of custody” refers to the set of procedures used to establish
an accurate written record that traces the possession of a sample from the moment it is
collected until it is analyzed in the laboratory. A chain of custody form is the specific
document used to record the possession and transfer of samples and ensure the integrity
of the samples from the time they are collected until data is reported. Each individual
involved in possession of the sample must sign a chain of custody form when the sample
is received or surrendered. Some general criteria included on a standard chain of
custody form include:
 Sample number
 Name and signature of sample collector/procurer/originator
 Date and time of sample collection/procurement/origination
 Place and address of sample collection/procurement/origination
 Sample type (clinical, environmental, etc.)
 Signature of persons involved in the chain of possession
 Inclusive dates of possession
UPDATE 3
In your discussions with staff in the microbiology/virology section, you are surprised and
concerned to discover that a laboratorian from the microbiology/virology section has
been out on sick leave for the past 2 days. You make some follow-up phone calls to
obtain additional information regarding the sick employee.
After further inquiry, you learn that the sick employee, a 45-year-old microbiologist
named Cathy Stevenson, has been hospitalized with several flu-like symptoms. Cathy
was among the few laboratory staff who had direct contact with the PT specimens, since
she was responsible for preparing the samples for analysis. Rapid influenza testing
reveals that she is indeed infected with influenza A, the same general type of flu as
H2N2. Confirmatory cultures required to determine the subtype of the strain will take
another 5-10 days.
12. Is shelter-in-place (workplace lockdown) a necessary and/or feasible action to take
in this situation? What measures are associated with sheltering-in-place? Who has the
authority to initiate a shelter-in-place order?
Suggested answer: Shelter-in-place is a control measure that is most appropriate in
situations in which there has been a release of a toxic chemical, biological agent, or
ionizing radiation into the air. In this case, sheltering-in-place would probably not be a
necessary or effective control measure. Given the routine safety precautions taken in the
laboratory setting, it is highly unlikely that staff members would be at any great risk of
acquiring H2N2 via the PT samples.
The following measures might be taken to shelter-in-place in an occupational setting:
 Close the business.
 If there are customers, clients, or visitors in the building, provide for their safety
by asking them to stay, not leave.
 Close and lock all windows, exterior doors, and any other openings to the outside.
If there is danger of explosion, close the window shades, blinds, or curtains.
 Turn on call-forwarding or alternative telephone answering systems or services.
If the business has voice mail or an automated attendant, change the recording to
indicate that the business is closed, and that staff and visitors are remaining in
the building until authorities advise that it is safe to leave.
 Have employees familiar with your building’s mechanical systems turn off all fans
and heating and air conditioning systems. Some systems automatically provide
for exchange of inside air with outside air; these systems, in particular, need to be
turned off, sealed, or disabled.
 Select an interior room(s) above the ground floor with the fewest windows or
vents and adequate space for everyone to be able to sit. Avoid overcrowding by
selecting several rooms if necessary. Large storage closets, utility rooms,
pantries, copy rooms, and conference rooms without exterior windows work well.
o Avoid selecting a room with mechanical equipment like ventilation blowers or
pipes, because this equipment may not be able to be sealed from the outdoors.
o It is ideal to have a hard-wired telephone in case you need to report a lifethreatening condition. Cellular telephone service may be overwhelmed or
damaged during an emergency.
 Gather essential disaster supplies, such as nonperishable food, bottled water,
battery-powered radios, first aid supplies, flashlights, batteries, duct tape, plastic
sheeting, and plastic garbage bags.
 Bring everyone into the room(s) and close and lock the door(s).
 Use duct tape and plastic sheeting (heavier than food wrap) to seal all cracks
around the door(s) and any vents into the room.
 Unless there is an imminent threat, ask employees, customers, clients, and visitors
to call emergency contacts to let them know where they are and that they are safe.
In this case, the state health department would most likely have the authority to mandate
a shelter-in-place order or any other isolation or quarantine order that might be
necessary for effective containment of an infectious agent.
UPDATE 4
As you attempt to maintain control of the situation in your state public health laboratory,
CAP has been in contact with the World Health Organization and the Centers for Disease
Control and Prevention via a continuous series of conference calls and emergency
meetings, and other communications.
It has been decided that the best way to handle the issue of the dangerous H2N2 shipment
is to mandate laboratories to destroy their PT samples via autoclaving or incineration as
quickly as possible. All laboratories in possession of H2N2 samples are instructed to
report back within 24 hours to ensure that all samples have been destroyed and properly
disposed of.
13. Knowing that you are dealing with H2N2, how will this affect the biosafety level
guidelines that should be followed for safe disposal of the samples?
Suggested answer: H2N2 strains of influenza are considered “non-contemporary”
human influenza and should be handled with increased caution, requiring biosafety level
3 (BSL-3) practices, procedures, and facilities. Laboratory staff involved in the
destruction of the samples should use PPE appropriate for a BSL-3 situation, such as
negative pressure HEPA-filtered respirators or positive air-purifying respirators
(PAPRs). Also be mindful of any clothing-related protocols for working with H2N2.
UPDATE 5
Confirmatory cultures from your ill laboratory employee, Cathy Stevenson, are finally
available. To the great relief of many, though Cathy is suffering from a severe bout of
Influenza A, it is not subtype H2N2. While it is promising to know that Cathy’s infection
was not found to be of the H2N2 strain, you do not yet consider yourself “out of the
woods.” As such, you continue to closely monitor other potentially-exposed laboratory
staff members for any signs or symptoms of flu.
Not surprisingly, given the widespread dissemination of the PT samples and the
involvement of the CDC and WHO, news of the dangerous PT shipment has hit the
media. Local and national newspapers and media outlets are covering the story. Given
the widespread national and international dissemination of the samples, the public
reaction is one of concern.
As expected, phones at your laboratory begin to ring off the hook with callers anxiously
awaiting answers. In addition, local hospitals, doctors’ offices, and emergency rooms are
observing an influx of “worried well” as a result of the news coverage and a leaked report
that a laboratory employee had been hospitalized with severe flu-like symptoms.
14. What communication mechanisms can be used to address the fears of the public and
properly communicate public health risks?
Suggested answer: Working in close collaboration with stakeholders, you should prepare
a message for responding to calls from the media, concerned citizens, etc. Your message
should include:
 A brief description of the incident and how it occurred
 A clear and honest explanation of the associated risks
 A clear explanation that Cathy’s influenza type A is NOT the H2N2 strain
 A plan of action that explains how the incident will be addressed
 A phone number, Web site, or other point of contact for those who want to learn
more
 A promise to keep the public updated as new information becomes available
CONCLUSION
You have managed, with the help of your highly trained laboratory staff, to safely and
successfully dispose of all of the dangerous H2N2 samples. On April 22, authorities
confirm that virtually all H2N2 samples have been accounted for and properly destroyed.
In addition, there are no reports of illness among laboratory staff or the general
population. For now, the crisis has been averted.
However, this issue raises concerns about the administration of PT programs, biosafety
procedures, and the ability of laboratories to respond to emergencies that involve
potential exposure to infectious agents.
NOTE: In April of 2005, the CAP administered a PT exercise to several public health laboratories located
in the United States and abroad. In an accident still not well-understood, the PT exercise resulted in
thousands of samples of improperly attenuated influenza A/H2N2 virus being mistakenly sent to
laboratories in 18 different countries. The samples were included in kits used regularly to test the ability of
laboratories to identify strains of the flu virus. Ninety percent of the laboratories that received test kits
were located in North America. Fortunately, all of the samples were destroyed, and there were no cases of
H2N2 reported in the affected populations
References
Centers for Disease Control and Prevention Advisory Committee on Immunization
Practices. Infection Control Measures for Preventing and Controlling Influenza
Transmission in Long-Term Care Facilities. 2007. Available at: http://0www.cdc.gov.mill1.sjlibrary.org/flu/professionals/infectioncontrol/ longtermcare.htm.
American Red Cross. Fact Sheet on Shelter-in-Place. 2003. Available at:
http://www.redcross.org/services/disaster/beprepared/shelterinplace.pdf.