Context Bioterrorist attacks involving letters and mail-handling systems in
Washington, DC, resulted in Bacillus anthracis (anthrax)
spore contamination in the Hart Senate Office Building and other facilities
in the US Capitol's vicinity.
Objective To provide information about the nature and extent of indoor secondary
aerosolization of B anthracis spores.
Design Stationary and personal air samples, surface dust, and swab samples
were collected under semiquiescent (minimal activities) and then simulated
active office conditions to estimate secondary aerosolization of B anthracis spores. Nominal size characteristics, airborne concentrations,
and surface contamination of B anthracis particles
(colony-forming units) were evaluated.
Results Viable B anthracis spores reaerosolized under
semiquiescent conditions, with a marked increase in reaerosolization during
simulated active office conditions. Increases were observed for B anthracis collected on open sheep blood agar plates (P<.001) and personal air monitors (P =
.01) during active office conditions. More than 80% of the B anthracis particles collected on stationary monitors were within
an alveolar respirable size range of 0.95 to 3.5 µm.
Conclusions Bacillus anthracis spores used in a recent
terrorist incident reaerosolized under common office activities. These findings
have important implications for appropriate respiratory protection, remediation,
and reoccupancy of contaminated office environments.
On October 15, 2001, a letter containing threatening language and a
light tan powdery substance was opened in the mail handling area of a Senate
office suite in the Hart Senate Office Building, Washington, DC. Federal officials
removed the letter and shut down the local air handling systems. The letter
was transported to the US Army Medical Research Institute of Infectious Disease
and was subsequently confirmed to contain viable Bacillus
anthracis (anthrax) spores that were dispersible in air.1 Scanning
electron microscopy of the spores used in the Senate office attack showed
that they ranged from individual particles to aggregates of 100 µm or
more. Spores were uniform in size and appearance and the aggregates had a
propensity to pulverize1 (ie, disperse into
smaller particles when disturbed).
Following the attack, nasal swabs were collected by other investigators
from more than 7000 building occupants and cultured for B anthracis. Twenty of 38 individuals in the office suite where the
envelope was opened had positive nasal swab tests including 13 individuals
present in the vicinity of the mail area and 7 workers on an interconnected
lower floor. Additionally, 2 workers from an adjacent office suite that entered
an adjoining contaminated hallway and 6 emergency responders who entered the
office or hallway had positive nasal swab tests.
The building was officially closed to the public on October 17, 2001,
with access to the contaminated suite limited to forensic investigators only.
This study was completed after forensic investigation and prior to remediation
of the Hart Senate Office Building.
Information regarding primary aerosolization of B
anthracis spores has been reported,2-5 but
few data are available regarding secondary aerosolization indoors. The purpose
of this investigation was to evaluate secondary aerosolization of viable B anthracis spores under both quiescent and active office
conditions. Understanding secondary aerosolization (reaerosolization) of B anthracis spores in building environments is essential
for exposure assessment and risk evaluation following bioterrorism attacks.
Such understanding will also guide cleanup strategies for readily dispersible
bioaerosols.
Environmental samples were collected in the affected Senate office suite
(total area approximately 1200 sq ft) beginning 25 days after the initial
incident. Stationary and personal air samples and surface samples were collected
during 3 separate building entries (Table
1). Initial semiquiescent sampling was followed by second and third
rounds of sampling under simulated active office conditions. All analyses
were conducted such that only viable spores or spore aggregates were recorded.
During semiquiescent sampling, movement was minimized in the suite while
air and surface samples were collected from various locations. During the
semiquiescent sampling, the sample team (wearing sterile gloves, boots, hooded
protective suits, and powered air purifying respirators with P.100 cartridges)
placed sampling devices in the locations indicated in Figure 1 and left the suite to reduce air turbulence for the duration
of the sample collection period. Following semiquiescent sampling, active
office conditions were simulated to reflect routine behaviors in a busy office
environment (ie, paper handling, active foot traffic, simulated mail sorting,
moving trash containers, patting chairs). There was no activity in the office
suite several days prior to or between sampling periods.
There are no validated environmental sampling or risk assessment methods
for B anthracis contamination. Questions regarding
collection techniques, laboratory extraction efficiency from environmental
media, and appropriate methods for air monitoring remain unanswered. Accordingly,
in this investigation a variety of environmental sampling methods were used
to assess their usefulness for estimating environmental exposure and risk
from B anthracis spores. Samples and sample locations
were based on plausible exposure pathways (both inhalation and dermal) and
were selected based on proximity to the original release, pedestrian traffic
patterns within the suite, representative exposures to the staff in the work
area, and areas of interest for spore transport within the office suite (eg,
computer monitors).
Environmental sampling methods included air monitoring with stationary
and personal sampling devices (devices worn by the sample team to characterize
colony-forming unit [CFU] levels in their breathing zone) that actively collected
spores from a known volume of air as well as open blood agar plates that passively
collected spores deposited from the Hart Senate Office Building aerosol. Surface
samples were collected to help characterize the presence of B anthracis contamination on a variety of surface types using both
microvacuum devices and sterile swabs. These environmental samples were collected
under both quiescent and active office conditions to assess the influence
of human movement within the suite on environmental spore concentrations.
Andersen 6-stage viable (microbial) particle-sizing samplers (Thermo-Andersen,
Smyrna, Ga) were used to collect airborne spores to evaluate concentrations
and size ranges of spores or spore aggregates. Andersen samplers were operated
for 10 minutes at an air flow rate of 28.3 L/min during each sample collection
period. The Andersen sampler collects spores according to nominal aerodynamic
diameters on each of 6 vertically stacked agar plates. Andersen samplers use
petri dishes filled with 42 mL of agar to control aerodynamics of particle
impact on plates according to manufacturer-specified cutoffs of 7.0, 4.7,
3.3, 2.1, 1.1, and 0.65 µm. For this investigation, 18 mL of 5% sheep
blood agar (SBA) plates (Remel Inc, Lenexa, Kan) were used for collection
media. Use of reduced media volume resulted in an increase in the specified
jet-to-plate distance of 0.3 cm with a corresponding increase of 0.3 µm
in the particle size cutpoints.6 Thus, the
smallest particle impacting the number 6 plate in the cascade would have a
nominal diameter of 0.95 µm (ie, 0.65 µm + 0.3 µm).
For the semiquiescent and the first active testing period, 2 viable
Andersen impact samplers (6-stage) were used; 1 was placed on the floor in
the vicinity of the original contamination and 1 was placed on the floor 20
feet away near the common entrance to the suite (Figure 1). During the second active sampling period, the 2 Andersen
samplers were placed at the breathing zone level in the same locations, and
a specially configured 2-stage Andersen sampler was placed at a floor location
near the original source zone. The final stage of this sampler was fitted
with a glass fiber filter to trap any remaining viable spores smaller than
the final impact stage (approximately 0.9 µm). At the end of each sample
collection period, Andersen samplers were disinfected to avoid cross-contamination.
Direct colony counts on SBA plates in the Anderson samplers were obtained
and the positive hole correction method
(Box) was used to acquire a statistical
probability count of CFUs (Table 2).
Box. Positive Hole Correction Method
The positive hole correction method determines a statistical probablility
count of colony-forming units. It represents a count of the jets that delivered
the spores to the agar plates and the conversion of the jet number to a particle
count by using the "positive hole" conversion formula
7:
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where Pr is the expected number of viable particulates to produce
r positive holes and N is the total number of holes per
stage (400). This formula is based on the principle that as the number of
viable particles being impinged on a given plate increases, the probability
of the next particle going into an unpenetrated hole decreases. Thus, when
9 of 10 of the holes have each received 1 or more particles, the next particle
has but 1 chance in 10 of going into an unpenetrated hole. Therefore, on average,
10 additional particles would be required to increase the number of positive holes by 1.
In addition to stationary air samples, personal air samples were collected
from the breathing zone of sample team members during all 3 rounds of sampling.
Sample pumps were calibrated to operate at a flow rate of 4 L/min. The flow
rate was not intended to simulate respiratory minute ventilation but to provide
efficient deposition of spores on the collection media. Collection media consisted
of gelatin filters placed in 37-mm open-faced filter cassettes and located
in breathing zones of team members for each sampling period. These cassettes
are commonly used for personal air monitoring applications and were available
with corresponding gelatin inserts conducive to the collection and direct
incubation of microbial samples. Sample cassettes were placed on the front
of the team members' suits just below the shoulder and connected to a sampling
pump worn at the waist by a length of Tygon tubing (Saint-Gobain Performance
Plastics Corporation, Akron, Ohio).
Open plates were placed in workstations, on the floor, and within the
stairway to estimate spore settling during and following various levels of
human activity in the suite. Seventeen SBA plates were placed in various locations
and at various heights throughout the office during the semiquiescent and
the first active sampling period. Ten plates were placed on office chairs,
3 at various floor locations, and 4 on the steps of an internal office stairway
(Figure 1). Plates were opened for
45 minutes to collect viable spores then closed and wrapped with parafilm.
A total of 17 surface samples were collected on fabric office dividers,
carpets, paper files, and near the source of the original contamination. A
microvacuum sampler was used to quantify the surface loading of B anthracis on a variety of surface types. Microvacuum samples were
collected using personal air monitoring pumps operated at a calibrated flow
rate of 4 L/min. Filter cowls containing gelatin filters with a nominal pore
size of 3 µm (having submicron retention efficiencies) were connected
to the pump with tubing to form a microvacuum device. Sampled areas were defined
by a 100-cm2 template, then vacuumed using a slow back and forth
motion first in one direction, and then perpendicular to the original direction.
Microvacuum samples were collected at workstations in 5 different office areas
during the second active sampling period.
Swab samples were used to assess the presence of B anthracis contamination on an additional 12 surfaces. Sterile nylon
swabs moistened with sterile water were used to sample both vertical and horizontal
surfaces as defined by 100-cm2 templates. Areas were swabbed in
perpendicular directions using a slowly progressing S-shaped motion and then
placed in sterile 15-mL tubes. Nine swab samples were collected for both the
semiquiescent and first active sampling periods: 3 vertical semigloss latex
painted surfaces (2 doors and 1 wall), 3 computer monitors, and 3 individual
mailboxes.
Aseptic handling techniques were used throughout the sampling and analytical
process. All samples were labeled immediately following collection using predetermined
sample codes. Samples were placed in individual resealable bags and immediately
shipped to the analytical laboratory with blind identification codes and under
chain-of-custody. Field blank samples (quality-control samples used to ensure
adherence to sterile microbiologic technique) were included at a frequency
of 10%.
Samples were evaluated for the presence of viable B anthracis at the Naval Medical Research Center in Silver Spring,
Md. Gelatin filters were removed from the filter cassettes and placed directly
on SBA plates. Swabs and glass fiber filters were macerated in 3.0 and 7.5
mL, respectively, of sterile phosphate-buffered saline for approximately 1
minute to free viable spores. Following maceration, a 1.0-mL aliquot of each
sample was removed and heat shocked at 65°C for 15 minutes to reduce viable
vegetative bacteria in the sample. A 200-µL aliquot of each heated sample
was spread on an SBA plate and plates were incubated at 37°C for 14 hours.
Following incubation, bacterial colonies morphologically consistent with B anthracis were counted and recorded. Rapid real-time
polymerase chain reaction assays were used to confirm the identity of suspect B anthracis colonies.8,9 At
least 1 suspect colony from each plate was tested for the presence of the
genetic markers pag and cyaB,
specific to the virulence plasmids pXO1 and pXO2, respectively. Following
polymerase chain reaction confirmation of selected suspect colonies, the number
of B anthracis colonies on each plate was reported.
Analyse-It Software version 1.64 (Analyse-It Software Ltd, Leeds, England)
was used for statistical analyses and P<.05 was
considered significant. All sample team members were specially trained in
response to extremely hazardous environments and all participation was voluntary.
The US Federal Incident Command System reviewed and approved the study. Incident
Command System is a system used to organize and manage participating groups
during emergency response situations.
Results for the 6-stage Andersen air samples are presented in Table 2. Positive hole correction results
are presented below where applicable. Comparison of floor samples between
semiquiescent and active conditions showed an increase in viable spore collection
across all sampler stages at both the mail area (48 vs >3006 total CFUs) and
entrance area (71 vs 204 total CFUs) locations. In the mail area, stationary
Andersen breathing zone samples showed an increase compared with semiquiescent
sampling taken previously at floor level (200 vs 48 total CFUs). Estimated
airborne spore concentrations collected near the floor over a 10-minute period
ranged from 171 to 251 CFUs/m3 during the semiquiescent period.
For the active period, airborne CFU concentrations ranged from 721 to more
than 11 000 and 106 to 707 CFUs/m3 for floor and breathing
zone samples, respectively. This represents as much as a 65-fold increase
in CFUs under active conditions compared with semiquiescent conditions. Approximately
half of the CFUs had corrected nominal diameters ranging from 1.4 to 2.4 µm,
with more than 80% ranging from 0.95 to 3.5 µm. Results from the 2-stage
Andersen sampler indicated no viable spores less than a corrected nominal
diameter of 0.95 µm.
Locations and results of viable colony counts on the 17 open SBA plates
(10 on chairs; 7 on the floor) collected during semiquiescent and active periods
are shown in Figure 1. During the
semiquiescent period, 5 of the 17 plates were positive for B anthracis (median, 0 CFU; range, 1-3 CFUs; 95% confidence interval
[CI], 0-1). In comparison, 14 of 15 plates (1 plate was left in the suite
and was desiccated beyond use) during the first active sampling period were
positive for B anthracis (median, 15 CFUs; range,
4-80 CFUs; 95% CI, 11-28) illustrating a significant increase in colony counts
(P<.001; using a 2-tailed nonparametric Wilcoxon
signed rank test).
Results of personal air monitor samples collected from team members
during each of the sampling periods are presented in Table 3. Filters from all 10 of the samples were positive for B anthracis. Results were positive for B anthracis during semiquiescent office conditions (mean, 4 CFUs; range,
1-7 CFUs) and increased during active office conditions (mean, 14 CFUs; range,
1-36 CFUs). There was a significant increase in the number of CFUs collected
on personal air samples during the second active test period (P = .01; 1-tailed paired t test with 2 df) but not the first active test period (P = .17) when compared with the semiquiescent sampling period. A 1-tailed
statistical test was used with the expectation that the number of airborne
viable CFUs would increase (rather than decrease) when activity increased
in the suite.
Six of the 9 surface swab samples taken during the semiquiescent and
first active period were positive; 3 vertical mailbox surfaces (range, 3-43
CFUs) and 3 computer screens (range, 2-150 CFUs), with little change in viable
spore counts in response to increased activity. Three swab samples collected
from vertical wall surfaces during each sampling period were negative. During
the second active sampling period, sequential swab samples of a computer monitor
screen sampled in the off, then on position, resulted in a 25-fold increase
in viable colony counts on the charged screen. Deposition of spores on the
charged monitor may indicate influence of electrostatic effects on spore behavior.
Additionally, 5 microvacuum samples were taken in different office areas
during the second period of activity to evaluate contamination of different
types of surfaces (Table 4). Although
microvacuum samples showed substantial viable spore contamination of carpeted
and smooth horizontal surfaces, very little contamination of vertical fabric
workstation dividers or the tops of paper files was found. No CFUs were found
on the field blanks collected from any of the sample types during the course
of the investigation.
The importance of secondary aerosolization of B anthracis spores associated with a bioterrorism attack has been discussed by
a number of researchers.10-14 However,
few empirical data existed to allow for scientifically based public health
conclusions or recommendations. Although research conducted by the military
has shown that Bacillus subtilis spores, used as
a surrogate for B anthracis, can reaerosolize with
varying activities in outdoor environments,13,15 until
now, no published data have been available concerning secondary aerosolization
of B anthracis spores indoors. Prior to the attacks
in the fall of 2001, consensus recommendations from the Working Group on Civilian
Biodefense11 suggested only a slight risk of
acquiring inhalational anthrax by secondary reaerosolization from heavily
contaminated surfaces. These recommendations were based on an incident involving
accidental release of B anthracis in Sverdlovsk,
Russia,5 occupational studies of workers in
goat hair processing mills,16 and modeling
analyses by the US Army.12 The Working Group
on Civilian Biodefense recognized that its recommendations were based on interpretation
and extrapolation from an incomplete knowledge base and needed to be regularly
reassessed as new information becomes available.11 A
recent reassessment by the consensus group includes a precautionary note regarding
reaerosolization of B anthracis spores based, in
part, on work presented here.17
This investigation presents empirical findings concerning secondary
aerosolization of viable B anthracis spores following
a bioterrorism incident indoors. Among the limitations of the work are the
severe schedule constraints, limited availability of equipment, and the extreme
conditions under which the investigation was planned and implemented. Both
empirically observed and substantially increased spore concentrations were
recorded on open SBA plates during active conditions in the office suite.
Elevations of CFUs recorded on personal air monitoring devices during active
vs semiquiescent office conditions are consistent with military investigations
showing activity-related increases in airborne spore exposures outdoors.13 However, the personal air monitor data reported in
this study are limited due to high variability and small sample size.
During simulated activities, airborne concentrations of viable B anthracis spores within the office ranged from 2 to 86
CFUs/m3 for personal air monitors and 100 to more than 11 000
CFUs/m3 for stationary Andersen samples, with more than 80% of
the spores falling into the respirable range (<5 µm). Relatively
higher collection efficiencies on stationary monitors may be due to sample
locations within the contaminated suite, higher air flow rates through the
stationary sampling devices, or the sample team personal monitors integrating
exposure over both contaminated and noncontaminated areas of the Hart Senate
Office Building (personal monitors were activated on entry to the building
6 floors below the contaminated suite).
Using a mean (SD) respiratory rate of 1.38 m3/h (0.66) reported
for office workers,18 estimated inhalation
exposures to B anthracis in the breathing zone were
119 and 250 CFUs/h for personal air monitors and breathing zone Andersen samplers,
respectively. Based on CFU concentrations recorded by floor level Andersen
samplers, estimated exposures were as high as 15 000 CFUs/h. Additionally,
findings of airborne B anthracis spores during the
initial semiquiescent sampling period suggest that even minimal movements
may result in resuspension of viable spores. These findings were recorded
almost a month following the original incident, despite the removal of the
contaminated letter from the suite.
Determining the magnitude of inhalational risks from reaerosolized B anthracis spores is uncertain. Reliable human data on
the minimum infective dose for inhalational B anthracis is lacking. Individual susceptibility, virulence of the strain, and
spore physical characteristics may all have profound impacts on the dose necessary
to cause inhalational anthrax.3,4 Primate
model extrapolations suggest an estimated human median lethal dose between
2500 and 55 000 spores,10 with the highest
infectivity associated with clouds of single spores, vs multispore aggregates.4 Recent primate studies have demonstrated inhalational
infectivity of B anthracis following exposure to
only a few spores.19 Human cases of inhalational
anthrax have also been reported involving minimal exposures.16 Risk
predictions indicate that infective doses may be as low as 1 to 3 spores14 and these predictions may be reflected in the 2 cases
of inhalational anthrax in New York and Connecticut still under investigation.20
This work clearly demonstrates a potential for secondary aerosolization
of viable B anthracis spores originating from contaminated
surfaces in an indoor environment. As a result, precautions to protect exposed
decontamination workers and area occupants are indicated.
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