Implications of Limits of Detection of Various Methods for Bacillus

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ImplicationsofLimitsofDetectionofVarious

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RiskstoHumanHealth

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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 2009, p. 6331–6339 Vol. 75, No. 19

0099-2240/09/$08.00⫹0 doi:10.1128/AEM.00288-09

Implications of Limits of Detection of Various Methods for

Bacillus anthracis in Computing Risks to Human Health

䌤

†

Amanda B. Herzog,

1,4

S. Devin McLennan,

Alok K. Pandey,

1,4

Charles P. Gerba,

4,6

Charles N. Haas,

4,5

Joan B. Rose,

3,4

and Syed A. Hashsham

1,2

Department of Civil and Environmental Engineering,

Center for Microbial Ecology,

Department of Fisheries and Wildlife,

and

Center for Advancing Microbial Risk Assessment,

Michigan State University, East Lansing, Michigan; Department of Civil,

Architectural and Environmental Engineering, Drexel University, Philadelphia, Pennsylvania

; and Department of Soil,

Water and Environmental Science, University of Arizona, Tucson, Arizona

Received 4 February 2009/Accepted 23 July 2009

Used for decades for biological warfare, Bacillus anthracis (category A agent) has proven to be highly stable

and lethal. Quantitative risk assessment modeling requires descriptive statistics of the limit of detection to

assist in deﬁning the exposure. Furthermore, the sensitivities of various detection methods in environmental

matrices are vital information for ﬁrst responders. A literature review of peer-reviewed journal articles related

to methods for detection of B. anthracis was undertaken. Articles focused on the development or evaluation of

various detection approaches, such as PCR, real-time PCR, immunoassay, etc. Real-time PCR and PCR were

the most sensitive methods for the detection of B. anthracis, with median instrument limits of detection of 430

and 440 cells/ml, respectively. There were very few peer-reviewed articles on the detection methods for B.

anthracis in the environment. The most sensitive limits of detection for the environmental samples were 0.1

CFU/g for soil using PCR-enzyme-linked immunosorbent assay (ELISA), 17 CFU/liter for air using an

ELISA-biochip system, 1 CFU/liter for water using cultivation, and 1 CFU/cm

for stainless steel fomites using

cultivation. An exponential dose-response model for the inhalation of B. anthracis estimates of risk at concen-

trations equal to the environmental limit of detection determined the probability of death if untreated to be as

high as 0.520. Though more data on the environmental limit of detection would improve the assumptions made

for the risk assessment, this study’s quantiﬁcation of the risk posed by current limitations in the knowledge of

detection methods should be considered when employing those methods in environmental monitoring and

cleanup strategies.

According to the Centers for Disease Control (CDC), a

category A agent is an organism that poses a risk to national

security because it can be easily disseminated or transmitted

from person to person, results in high mortality rates, has the

potential for major public health impact, might cause public

panic and social disruption, and requires special action for

public health preparedness (http://emergency.cdc.gov/agent

/agentlist-category.asp). Quantitative information on category

A agents in environmental matrices (soil, air, fomite, and wa-

ter) is very limited (62). However, from the literature, it has

been concluded that Bacillus anthracis is the most environmen-

tally stable category A agent overall (62).

After the release of B. anthracis through mail envelopes in

2001, assessment of the decontamination process revealed an

important question: could the detection methods effectively

determine if the environment is clean? An evaluation of the

effectiveness of sampling methods at a U.S. postal facility in

Washington, DC, that was contaminated with B. anthracis

spores concluded that neither of the sampling methods used

(HEPA vacuum or wipes) were sensitive enough to ensure that

spores had been removed completely. In addition, the event

exposed the necessity of quantifying recovery and extraction

efﬁciency during sample collection and processing to improve

the method limit of detection (61, 67).

In this literature review, the limit of detection of methods for

B. anthracis is characterized as either an instrument limit of

detection or an environmental limit of detection. An instru-

ment limit of detection is generally evaluated with pure cul-

tures. An environmental limit of detection is evaluated with

cultures/cells spiked into an environmental matrix (soil, air,

fomites, water), which then undergoes various recovery and

concentration procedures (i.e., ﬁltration and extraction or di-

rect extraction) before detection (see Fig. S1 in the supple-

mental material).

Compared to an instrument limit of detection, the establish-

ment of an environmental limit of detection poses more chal-

lenges, including dilute target concentrations, environmental

impurities, background inhibitors, organisms in a viable but

not cultivable state, and overall processing efﬁciency. There are

many steps for processing environmental samples prior to de-

tection. At each process step, there can be a loss of the initial

target organism, and thus, each step has a recovery efﬁciency,

which could be interpreted as a set number, distribution, or

range (see Fig. S1 in the supplemental material). Since recov-

ery efﬁciency directly affects the limit of detection, improving

recovery efﬁciency would result in a more sensitive detection

method.

* Corresponding author. Mailing address: Department of Civil and

Environmental Engineering, A126 Research Complex—Engineering,

East Lansing, MI 48824. Phone: (517) 355-8241. Fax: (517) 355-0250.

E-mail: [email protected].

† Supplemental material for this article may be found at http://aem

.asm.org/.

䌤

Published ahead of print on 31 July 2009.

6331

In determining if an environmental site is “clean,” another

component that should be evaluated is the quantiﬁcation and

characterization of the potential health risk. Quantitative mi-

crobial risk assessment (QMRA) is a method used to assess the

likelihood of infection based on speciﬁc exposures to hazard-

ous pathogenic organisms. QMRA risk modeling has been

used with water and food and could be useful for management

decisions during a disease outbreak or a bioterrorism attack

(35). Environmental monitoring is used to inform the exposure

assessment and the efﬁciency of disinfection. The limit of de-

tection is a critical criterion for any method, which dictates the

application and usefulness of demonstrating a “zero” during

environmental monitoring. The limit of detection of a chosen

analytical method is also an input variable for the QMRA

model; a statistical distribution quantifying the variability in

limit of detection is preferred for realistic modeling.

The objectives of this study were to review, in the literature,

the instrument limit of detection and the environmental limit

of detection for methods to detect B. anthracis and to compare

the estimated risk at the instrument limit of detection and the

environmental limit of detection. Though the number of arti-

cles on B. anthracis was extensive, there was a paucity of arti-

cles that speciﬁcally included environmental limits of detec-

tion. This information is essential for a QMRA of B. anthracis

in the establishment of future environmental monitoring strat-

egies and cleanup goals.

MATERIALS AND METHODS

Journal articles were searched on the ISI Web of Science database for B.

anthracis and the following keywords: method, sensitivity, limit of detection,

detection limit, limit, water, air, soil, fomite, surface, speciﬁcity, PCR sensor,

environmental, rapid, assay, diagnostic, immunoassay, antibody, real time, real-

time PCR, microﬂuidic, polymerase, quantitative, bioaerosol, aerosol, microar-

rays, biosensor, electrochemiluminescence (ECL), Raman spectrometry, and

mass spectrometry. Approximately 1,700 references (and abstracts, when avail-

able) were retrieved and were saved in an EndNote ﬁle. Though the search

defaults were set for the years 1900 through 2007, the oldest article used to

evaluate the limit of detection was published in 1994. Abstracts were manually

screened for information on the detection of B. anthracis. Some studies used a

surrogate for B. anthracis to determine the limit of detection. It was assumed that

B. anthracis would behave as the surrogate and was included in this review. If the

abstract pertained to a detection method, then the full article was downloaded,

saved in another database, and reviewed for quantitative data describing the limit

of detection. The remaining references and abstracts that were not used in this

literature review either did not indicate information about detection methods or

were not retrievable. At the end, 71 articles were retrieved and analyzed to

obtain the instrument limit of detection or the environmental limit of detection.

Instrument limit of detection. The instrument limit of detection was extracted

from the articles describing a method that detected B. anthracis in a pure culture

without spiking B. anthracis into an environmental matrix (soil, air, fomite, or

water). Raw data that were extracted were recorded in numbers of units of cells,

spores, DNA, CFU, protective antigens, and genomic copies in volumes that

ranged from liters to microliters. Articles that used units of protective antigens

were not used in this literature review due to the unknown conversion factor

from antigens to cells. All data were converted into standard units of cells per

milliliter of reaction solution, and the data by method were graphed and com-

pared.

Environmental limit of detection. In studies reporting the environmental limit

of detection, B. anthracis spores were spiked into the matrix, extracted, and

detected using various detection methods. The articles that reported the envi-

ronmental limit of detection of B. anthracis were categorized according to the

matrix in which B. anthracis was detected (soil, air, fomite, or water). Additional

parameters extracted from the articles varied with the matrix (see Fig. S2 in the

supplemental material). These included the following parameters. (i) For soil,

they include the amount of soil, sample concentration, extraction volume, vol-

ume of extracted sample added to the reaction, and total volume. In addition, the

type of pretreatment or extraction method and the soil type or location were

noted (Table 1). (ii) For air, they include the sample volume, airﬂow rate,

duration, sample concentration, extraction volume, volume of extracted sample

added to the reaction, and total volume. (iii) For fomites, they include the

surface area, sample concentration, surface seeding method, extraction volume,

and total volume. In some cases, recovery efﬁciency and extraction efﬁciency

were available and noted. In addition, the type of fomite, sampling method,

extraction method, and culturing method were noted (Table 2). (iv) For water,

they included the sample volume, sample concentration, extraction volume,

volume of extracted sample added to the reaction, and total volume. In addition,

the condition of the water was noted.

Quantifying limits of risk estimates. The risk of mortality by inhalation of B.

anthracis spores was estimated for concentrations corresponding to the instru-

ment limit of detection and the environmental limit of detection in the air. For

each limit of detection, a distribution of risks was calculated by the Monte Carlo

method using 100,000 replicates in Crystal Ball 7.3.1 (2007; Oracle). The number

of replicates was chosen at the point where the 90% conﬁdence interval was

stable over a range from 1/10 to 10 times the number of replicates used.

A recent evaluation of dose-response data for B. anthracis spores through the

inhalation exposure route found that the dose-response relationship could be

modeled by the exponential equation (4)

P共d兲 ⫽ 1 ⫺ e

⫺kd

where P(d) is the probability of death (P) (when untreated) at dose d, and k is the

probability that one organism will survive to initiate the response (4). In this

study, a k value generated from a pooled guinea pig and rhesus monkey data set

was used. A distribution of 10,000 best-ﬁt k values generated using bootstrap

replicates of that data set was provided by Timothy Bartrand of Drexel Univer-

sity and ﬁt to a gamma distribution. The dose was calculated as

d ⫽ C

air

䡠 R 䡠 t

where C

air

is the number of spores per cubic meter of air (instrument limit of

detection or environmental limit of detection), R is the breathing rate (m

/h),

and t is the duration of exposure (h). When C

air

was evaluated as a range of limits

of detection, it was modeled as a lognormal distribution; otherwise, it was

evaluated as a point estimate. The breathing rate, R, was modeled as a Pareto

distribution ﬁt to the short-term breathing rates of adults (18 years of age and up)

of both sexes from rest to moderate activity (71). The exposure time, t, was

modeled as a uniform distribution from 1 min to 8 h.

Five risk scenarios were evaluated with this model using different values for

air

. For each risk scenario, either the instrument limit of detection or environ

mental limit of detection, a sensitivity analysis was generated using Crystal Ball

7.3.1 (2007; Oracle). The median real-time PCR instrument limit of detection

and the range of real-time PCR instrument limit of detection were two scenarios

used to explore the effect of instrument limit of detection on risk. For the

instrument limit of detection scenarios, it was assume that all B. anthracis spores

in a cubic meter of air could be collected without any loss and concentrated into

1 ml of solution for analysis. Log-transformed real-time PCR and PCR instru-

ment limits of detection were checked for normality with a Lilliefors test and

compared using analysis of variance. Then, the range of PCR instrument limits

of detection was combined with the range of real-time PCR instrument limits of

detection to increase the data in the distribution.

There were three environmental limit of detection scenarios; C

air

was set to

the environmental limits of detection reported for B. anthracis detected in the

air. There were only two articles on the environmental limit of detection in the

air. Due to the lack of data on the environmental limit of detection, the two limits

of detection were referred to as the lower and upper environmental limits of

detection. These two risk scenarios were evaluated as point estimates. The last

risk scenario assumed that the environmental limit of detection for the air ﬁt the

same distributions as the lognormal instrument limit of detection, ranging from

17,000 to 50,000 CFU/m

(this may not be the true range).

RESULTS AND DISCUSSION

Instrument limit of detection. Out of 56 articles on the

instrument limit of detection, 17 articles were on real-time

PCR (6, 7, 11, 23, 25, 27, 39, 41, 45, 49, 51, 53, 56, 58, 60, 70,

72), 6 were on PCR (13, 31, 48, 57, 76), 10 were on biosensors

(1, 3, 21, 22, 33, 36, 40, 52, 73, 74), 5 were on microarray/PCR

(5, 19, 50, 64, 75), 6 were on immunoassay (29, 30, 32, 46, 65,

6332 HERZOG ET AL. APPL.ENVIRON.MICROBIOL.

TABLE 1. Parameters for the environmental limit of detection in soil

Detection method

Amt of soil Sample concn

Pretreatment/extraction

method (company)

Time

(h)

Difﬁculty

level

Extraction

vol (␮l)

Vol

added

reaction

(␮l)

Total

vol

(␮l)

Limit of

detection

(CFU/g

soil)

Soil type/location Reference

PCR-ELISA 100 g 1–100 CFU/100 g Easy DNA kit (Invitrogen) 2.5 2 100 60 60 0.1 Nonsuspicious sites 10

100 g 1–100 CFU/100 g Easy DNA kit (Invitrogen) 2.5 2 100 60 60 1.0 Contaminated sites with

organic compounds and

tanning agents

Nested PCR ⫹ 2⫻

cultivation in TSB

1 g 0, 1, 10, 10

,10

CFU/g

FastDNA SPIN kit 36.0 4 25 1.0 Garden soil with 3% peat 24

Nested PCR ⫹

cultivation in TSB

1 g 0, 1, 10, 10

,10

CFU/g

FastDNA SPIN kit 18 3 25 1.0 ⫻ 10

Garden soil with 3% peat 24

Nested PCR 1 g 0, 1, 10, 10

,10

CFU/g

FastDNA SPIN kit 2 2 25 1.0 ⫻ 10

Garden soil with 3% peat 24

100 mg 10

CFU/100 mg

Three freeze-thaw cycles/

glass beads and glass

milk

3.5 5 30 5 25 1.0 ⫻ 10

Litter, meadow, cultivated,

swamp, and lawn

PCR 1 g 2.5 ⫻ 10

–2.5 ⫻ 10

CFU/g

Hot detergent/bead mill

homogenization

1 3 100 10 100 2.5 ⫻ 10

Anthony ﬁne sandy loam from

New Mexico agriculture

ﬁelds

Immunoﬂuorescence 1 g 10

–10

CFU/g

Aqueous polymer two-

phase system

0.75 2 100 20 40 5.6 ⫻ 10

Sand 2

1g 10

–10

CFU/g

Aqueous polymer two-

phase system

0.75 2 100 20 40 1.4 ⫻ 10

Garden 2

Real-time PCR 0.1 g 10

–10

CFU/g

Heat treatment with 1.22

g/ml sucrose-0.5%

Triton X-100

0.75 3 1,000 5 25 1.0 ⫻ 10

National Institute of Health—

Korea

Multiplex PCR 0.1 g 10

–10

CFU/g

Heat treatment with 1.22

g/ml sucrose-0.5%

Triton X-100

0.75 3 1,000 1 25 1.0 ⫻ 10

National Institute of Health—

Korea

0.1 g 10

–10

CFU/g

Heat treatment with

sterilized water and 10%

Triton X-100-PBS

1.5 3 1,000 1 25 1.0 ⫻ 10

National Institute of Health—

Korea

IM-ECL 1 mg 0–10

CFU/assay

IM separation performed

twice and resuspended

in PBS

1.5 3 1.0 ⫻ 10

Moist, dark brown to black soil

and dry, light yellowish

sandy soil from diverse

military and agriculture

ﬁelds

1 mg 0–10

CFU/assay

IM separation performed

twice and resuspended

in PBS

1.5 3 1.0 ⫻ 10

Moist, dark brown to black soil

and dry, light yellowish

sandy soil from diverse

military and agriculture

ﬁelds

1 mg 0–10

CFU/assay

IM separation performed

twice and resuspended

in PBS

1.5 3 1.0 ⫻ 10

Moist, dark brown to black soil

and dry, light yellowish

sandy soil from diverse

military and agriculture

ﬁelds

Biosensor assay 1 mg/ml powder

in PBS

3.2 ⫻ 10

–3.2 ⫻ 10

CFU/ml

Washing with 1 ml PBST 0.25 1 30 5 25 3.2 ⫻ 10

Talc-based powder, cornstarch,

confectioners’ sugar, baking

soda, and Bacillus

thuringiensis-based pesticide

TSB, Trypticase soy broth.

VOL. 75, 2009 LIMITS OF DETECTION OF METHODS FOR B. ANTHRACIS 6333

68), 3 were on ECL (17, 34, 77), 2 were on enzyme-linked

immunosorbent assay (ELISA) (12, 26), 3 were on Raman

spectroscopy (37, 55, 78), and 4 were on mass spectrometry (8,

9, 28, 43) (Fig. 1). Limits of detection ranged from 10 cells/ml

(for real-time PCR) to 10

cells/ml (for mass spectrometry).

Considering the median instrument limit of detection, real-

time PCR and PCR were the most sensitive methods, with

median instrument limits of detection of 430 and 440 cells/ml,

respectively. It should be noted that there was one instrument

limit of detection (4.29 ⫻ 10

cells/ml) that was not added to

the distribution for real-time PCR because it was a multiplex

assay, and the other instrument limits of detection in the dis-

tribution were from a singleplex assay (42). The least-sensitive

methods were Raman spectroscopy and mass spectrometry,

with median instrument limits of detection of approximately

1.0 ⫻ 10

and 8.0 ⫻ 10

cells/ml, respectively.

The number of journal articles on real-time PCR and bio-

sensors allowed limits of detection to be ﬁt to a statistical

distribution. When fewer articles were published, as was true

for the other eight methods, assigning distributions was not

possible. ECL, ELISA, Raman spectroscopy, and mass spec-

trometry (having less than four articles) were the methods with

the least-sensitive instrument limits of detection. With limited

information on these methods, the median instrument limit of

detection may not properly represent these detection methods’

capabilities for detecting B. anthracis. For example, the instru-

ment limit of detection for ECL had only three published

articles, with limits of detection ranging from 10

cells/ml to

cells/ml. For some emerging techniques, such as immuno

magnetic ECL (IM-ECL) and aptamer-magnetic bead-ECL,

limits of detection differed by 4 orders of magnitude. While the

instrument limit of detection gives insight into the instruments’

capabilities, when evaluating cleanup goals and assessing risk,

the environmental limits of detection are needed to under-

stand the challenges and capabilities for addressing the con-

tamination.

Environmental limit of detection. Out of 15 articles on the

environmental limit of detection, 8 articles were on detection

in soil (2, 10, 18, 24, 44, 60, 63, 69), 2 were on detection in the

air (47, 66), 6 were on detection on fomites (14, 15, 16, 20, 38,

59), and 1 was on detection in water (54). The results for the

environmental limit of detection could not be reported as

distributions due to the limited number of articles for each

matrix. The two most predominant methods used for the en-

vironmental limit of detection were cultivation and PCR-based

methods.

Soil. The environmental limit of detection of B. anthracis

spiked into soil ranged from 0.1 (reported as 10 CFU/100 g of

soil) to 3.2 ⫻ 10

CFU/g of soil, with a median limit of detec

tion of 1.2 ⫻ 10

CFU/g of soil (Table 1). The median envi

ronmental limit of detection for soil should be used with cau-

tion, since there is a 9-orders-of-magnitude range due to the

many approaches used to evaluate the environmental limit of

detection. The approximate time for the extraction method

(Table 1) was the time for one sample to be processed based

on the information reported. If it was not an automated ex-

traction procedure, then with the increase in samples, there

would be an increase in extraction process time. The difﬁculty

level for the extraction process (1 to 5, easy to difﬁcult) was

based on the number of steps in the procedure, the preparation

time, and the approximated time for the extraction (Table 1).

The biosensor assay, the easiest extraction method, resulted in

the poorest limit of detection (3.2 ⫻ 10

CFU/g of soil). The

detection methods with the most-sensitive limits of detection

(PCR-ELISA, nested PCR, and PCR) had extraction methods

with difﬁculty levels ranging from 2 to 5 (Table 1).

The environmental limit of detection depended highly on

the pretreatment/extraction process; for instance, in 2003, Ryu

et al. (60) used multiplex PCR and reported a difference of 3

orders of magnitude between heat treatment with 1.22 g/ml

sucrose–0.5% Triton X-100 and heat treatment with sterilized

water and 10% Triton X-100–phosphate-buffered saline (PBS)

(Table 1). Similar results were found by Bruno and Yu in 1996

(18) when using IM-ECL as the detection method.

Differences in the environmental limit of detection were also

based on the location or the type of soil. In 1999, Beyer et al.

(10) reported that the PCR-ELISA method was more sensitive

when using soils from nonsuspicious locations compared to

TABLE 2. Parameters for the environmental limit of detection on fomites

Study authors

(reference)

Surface

area

Sample concn Surface seeding Sampling method Extraction method

Hodges et al. (38) 10 cm

0.2–3,000 CFU/cm

Inoculated with 0.5 ml spore

solution

Macrofoam swab Vortex in 5 ml PBST for 2 min

at 10-s intervals

Rose et al. (59) 25 cm

2 ⫻ 10

CFU/cm

Inoculated with 0.5 ml spore

solution

Cotton swab,

macrofoam swab

Vortex in 5 ml PBST for 2 min

at 10-s intervals

Brown et al. (15) 25 cm

100–10,000 CFU/cm

Dry aerosol deposition Polyester-rayon blend

gauze wipe

Sonication and heat treatment

Brown et al. (16) 25 cm

100–10,000 CFU/cm

Dry aerosol deposition Rayon swab Sonication and heat treatment

Brown et al. (14) 100 cm

100–10,000 CFU/cm

Dry aerosol deposition Vacuum ﬁlter sock Sonication and heat treatment

Buttner et al. (20) 1 m

CFU/m

Inoculated with spore solution BiSKit—wet/dry Foam compression

The limit of detection was not recorded in the article and was calculated to be at least 20 CFU/cm

6334 HERZOG ET AL. APPL.ENVIRON.MICROBIOL.

using those from former tannery sites. Agarwal et al. (2002) (2)

reported that the immunoﬂuorescence assay was more sensi-

tive when spores were spiked into sand (10

) rather than into

garden soil (10

). For the IM-ECL method, Bruno and Yu

(1996) (18) reported differences due to different strains, with

Sterne (10

) being more sensitive in the assay than Ames (10

)

and Vollum B1 (10

Air. There were only two studies of the evaluation of aero-

solized B. anthracis spores collected by an air sampler and

extracted for detection. The ELISA-biochip system coupled

with a portable bioaerosol collection system collected aerosol-

ized spores at an air sampling rate of 150 liters/min for 2 min

into 5 ml of PBS. The ELISA-biochip system consisted of an

ELISA for antibody-based identiﬁcation in combination with

the biochip detection instrument. The environmental limit of

detection of the ELISA-biochip system was 17 CFU/liter. For

the ELISA-biochip system, the efﬁciency of the air sampler was

reported as approximately 50%, but the distribution was not

fully described (66). The anthrax smoke detector collected

aerosolized spores using a bioaerosol collection system at a

rate of 15 liters/minute for 1 min onto a glass ﬁber ﬁlter tape.

The detection of the spores using the lifetime-gated ﬂuorime-

ter occurred after a thermal lysis and addition of TbCl

. The

environmental limit of detection of the anthrax smoke detector

was 50 CFU/liter (47).

Fomites. Spores were seeded on fomites (stainless steel,

plastic, wood, glass, etc.), recovered, extracted, and detected by

cultivation. The environmental limit of detection was evalu-

ated from stainless steel fomites ranging in surface area from

10 cm

to1m

(Table 2). In 2007, Brown et al. (14–16) also

evaluated the environmental limit of detection on painted wall-

board. In addition, the vacuum ﬁlter sock study tested porous

fomites, carpet, and concrete (14).

The sampling methods evaluated by the articles were use of

a macrofoam swab, cotton swab, polyester-rayon blend gauze

wipe, rayon swab, vacuum ﬁlter sock, and biological sampling

kit (BiSKit) (Table 2). Sampling methods such as use of cotton,

macrofoam, polyester, and rayon swabs were all tested by Rose

et al. in 2004 (59). It was concluded that the cotton and mac-

rofoam swabs produced the highest recovery when the swabs

were premoistened rather than dry. Similarly, in 2004, Buttner

et al. (20) tested the BiSKit, cotton swabs, and foam swabs.

The BiSKit was designed to do wet and dry sampling of large

surfaces for bacteria, viruses, and toxins. The BiSKit resulted

in the highest recovery out of the three methods. Using a

wetting agent to recover spores from the surfaces enhanced the

recovery and environmental limit of detection. Brown et al.

(2007) (15, 16) used sterilized deionized water (except when

using the vacuum ﬁlter sock), Buttner et al. (2004) (20) used

potassium phosphate buffer with 0.05% Tween 20, and the

other two authors used PBS with 0.04% Tween 80 (PBST).

FIG. 1. Distribution of the instrument limits of detection for vari-

ous methods. On each box plot, the solid line represents the median

result, and the dashed line represents the mean result. The box plot

whiskers above and below the box indicate the 90th and 10th percen-

tiles, respectively. The solid circles represent the outlying limits of

detection, and n represents the number of journal articles available on

each detection method for Bacillus anthracis.

TABLE 2—Continued

Extraction vol

Total

vol

Recovery efﬁciency (%)

Extraction

efﬁciency (%)

Culture medium Limit of detection

5 ml 31.7–49.1 93.4 Sheep blood agar 12 CFU/cm

5 ml 100 ␮l 41.7 for cotton swab, 43.6

for macrofoam swab

93.9 for cotton swab,

93.4 for

macrofoam swab

Trypticase soy agar with

5% sheep blood

20 CFU/cm

30 ml 1 ml 35 for stainless steel, 29

for painted wallboard

93 Brain heart infusion agar 90 CFU/cm

for stainless steel,

105 CFU/cm

for painted

wallboard

10 ml 1 ml 41 for stainless steel, 41

for painted wallboard

76 Brain heart infusion agar 1 CFU/cm

for stainless steel

and painted wallboard

30 ml 1 ml 29 for stainless steel, 25

for painted wallboard,

28 for carpet, 19 for

concrete

Petriﬁlm aerobic count

plate

105 CFU/m

for stainless steel

and carpet, 102 CFU/m

for

painted wallboard, 160

CFU/m

for concrete

3.3 ml for wet sampling,

16.1 ml for dry

sampling

1 ml 11.3 for wet sampling,

18.4 for dry sampling

Trypticase soy agar 42 ⫾ 6 CFU/m

for wet

sampling, 100 ⫾ 10 CFU/m

for dry sampling

OL. 75, 2009 LIMITS OF DETECTION OF METHODS FOR B. ANTHRACIS 6335

According to the CDC, the recommended wetting agents

were sterile water, a sterile saline solution, or a sterile phos-

phate-buffered solution (http://www.bt.cdc.gov/Agent/Anthrax

/environmental-sampling-apr2002.asp).

The detection method used for all fomite studies was culti-

vation; however, a different agar was used in each study. The

focus of the Rose et al. (2004) (59) article was achieving the

best recovery, and the study did not determine an environmen-

tal limit of detection. From the information given in the article,

the environmental limit of detection was calculated by using

the initial suspension concentration, the surface area, and the

lowest recovery reported. The calculated environmental limit

of detection was approximately 20 CFU/cm

The recovery efﬁciencies for all the fomite studies ranged

from 10 to 50%, and the extraction efﬁciencies ranged from 75

to 99%. Recovery of B. anthracis spores from fomites depends

on many parameters, such as fomite type, sampling procedure,

and sampling processing for detection. The recovery efﬁciency

from the sampling method was primarily the controlling factor

in determining the limit of detection and secondarily the efﬁ-

ciency from the extraction method.

Interestingly, in survival studies using cultivation as the de-

tection method on fomites, surface characteristics, relative hu-

midity, and temperature were the most important contributors

to viability (62). It was not clear whether recovery and limit of

detection changed with time in the environment, as this was

difﬁcult to differentiate from survival/degradation of the target.

However, this distinction could be made by adding a marker

along with the biological agent that does not degrade. For

environmental monitoring, the separate time dependence of

survival and recovery will be critical to deﬁne in future studies.

Only the articles from Brown et al. in 2007 (14–16) reported

and maintained the relative humidity and temperature in the

fomite studies at 30% ⫾ 10% and 25 ⫾ 2°C, respectively.

Determining and maintaining the relative humidity and tem-

perature that are most optimal for viability may increase re-

covery efﬁciency. In addition, this information could be used at

a contaminated site to inform ﬁrst responders of the possible

viability of remaining levels of the biological agent of concern.

Water. The spores were spiked into a volume of water,

ﬁltered through a 0.2- to 0.45-␮m-pore-size ﬁlter, extracted

from the ﬁlter, and then detected by various methods. The

main challenge for detection of B. anthracis in water was the

ability to concentrate the sample. If the sample was too dilute,

then the number of B. anthracis cells per liter of water could

fall below the environmental limit of detection. When the

sample is concentrated, some loss of the initial cells is likely.

There was only one article that evaluated the detection of B.

anthracis in water; the lack of articles could be due to this

matrix being less likely a vehicle for transmission (62). Perez et

al. (2005) (54) spiked B. anthracis spores into tap and source

water in volumes ranging from 0.1 to 10 liters. Sample concen-

trations were detected using sheep red blood cell agar plates,

B. anthracis chromogenic agar plates (R&F Laboratories),

PCR, or nested PCR.

Cultivation was used to determine the viability of the organ-

isms in the sample, and PCR was used to conﬁrm the identities

of any suspect colonies. When using the cultivation approach

for the source water samples (Chesapeake Bay and Patuxent

River), overgrowth of nontargeted ﬂora occurred in all studies.

PCR was only successful for testing source water when the

sample concentrations were at least 26 CFU/ml. The environ-

mental limit of detection for tap water was reported as 10

CFU/10 liters using the cultivation methods, while for PCR-

based methods, the environmental limit of detection decreased

to 534 CFU/liter. Though the PCR-based methods have a

rapid detection time compared to that of the cultivation meth-

ods (more than 24 h), in this case, PCR was less sensitive.

Challenges, such as loss of initial cells, could occur when con-

centrating large sample volumes (i.e., 10 liters) into 5 to 10 ␮l

for the PCRs.

Quantifying limits of risk estimates. Five risk scenarios us-

ing instrument limits of detection and environmental limits of

detection for C

air

were evaluated. Log-transformed PCR and

real-time PCR instrument limits of detection were normally

distributed (Lilliefors test, P of 0.65 for PCR, P of 0.78 for

real-time PCR) and were not signiﬁcantly different (analysis of

variance, P of 0.94). Therefore, the PCR and real-time PCR

instrument limit of detection distributions were combined to

increase the data set for the real-time PCR instrument limit of

detection. With the assumption of 100% recovery, the median

risk when C

air

equaled the median real-time PCR instrument

limit of detection was 0.006. When C

air

was modeled with a

lognormal distribution of real-time PCR instrument limits of

detection, the estimated risk was 0.0062. The median risk of

death from the inhalation of the entire dose of B. anthracis at

the environmental limit of detection in air was 0.22 at the lower

reported environmental limit of detection and 0.52 at the up-

per environmental limit of detection. Assuming that the envi-

ronmental limit of detection would have a similar distribution

as the instrument limit of detection (lognormal) and ranged

from 17,000 to 50,000 spores/m

, the median risk of death was

0.32 (Table 3). This assumption should be further evaluated

with environmental studies to conﬁrm that the environmental

limit of detection would have the same distribution as the

instrumental limit of detection. These risk estimates assumed

that 100% of the spore sample was inhalable. Risk estimates

were also reported for the percentages of 66.5%, 10%, and 1%

of spores in the sample that were inhalable or respirable (Table

3). Approximately 70% of inhaled air volume actually contacts

alveoli in the lungs, allowing spores to enter the body (71). In

addition, the 5th and 95th percentiles of each risk distribution

were used to deﬁne a 90% conﬁdence interval for each risk

estimate (Table 3).

A sensitivity analysis of the risk model was generated by

Crystal Ball 7.3.1 (2007; Oracle) for each of the ﬁve risk sce-

narios. For the real-time PCR instrument limit of detection

lognormal distribution, the limit of detection (79.4%) was the

most sensitive factor in determining risk, followed by the ex-

posure time (11.7%) and breathing rates (8.4%). The dose-

response function parameter k (0.5%) had the least impact on

the risk estimates. Similarly, for the assumed environmental

limit of detection lognormal distribution, the analysis resulted

in the exposure time (45%) being the most signiﬁcant factor,

followed by the limit of detection (27.5%), breathing rates

(26.1%), and the k parameter (0.9%). The median real-time

PCR instrument limit of detection and the two environmental

limit of detection (lower and upper) scenarios resulted with the

exposure time being the dominant factor in determining risk,

followed by breathing rates and the k parameter. C

air

values in

6336 HERZOG ET AL. APPL.ENVIRON.MICROBIOL.

these scenarios are point estimates rather than a distribution;

therefore, the limit of detection was not a measured parameter

in the sensitivity analysis.

Even assuming perfect sample collection and processing (no

loss in initial concentration), the estimated risk at the instru-

ment limit of detection was far above the commonly used

1:10,000 level. Environmental limits of detection increase due

to the imperfect efﬁciency of sample collection and processing,

increasing the risk at these higher detectable concentrations.

These risk estimates show that, using current techniques re-

ported in the literature, even allowing for all possible improve-

ments in collection technology, any detectable B. anthracis

constitutes an unacceptable risk. Moreover, these estimates

deﬁne the lowest risk that could be determined from measure-

ment, quantifying the risk that can exist even when no B.

anthracis was detected.

Finding signiﬁcant risk at B. anthracis limits of detection

suggests that direct measurement will rarely be adequate for

declaring a contaminated site as “clean,” and alternative ap-

proaches (e.g., extrapolating from demonstrated log reduc-

tions) are needed. For fomites, soil, and water, further work is

needed regarding the probability of infection by ingestion and

contact before one can adequately address limits of detection

and risk estimates. Direct measurement could, at best, reveal a

catastrophic failure of decontamination. With respect to pre-

ventative monitoring, these estimates showed that signiﬁcant

risk was posed by undetectable concentrations of B. anthracis

spores. This means that a low-concentration B. anthracis re-

lease would be more likely to be detected by the symptoms in

exposed humans rather than by current sampling technology.

Where there was danger or suspicion of a B. anthracis release,

close monitoring of human health would be needed, in addi-

tion to environmental sampling in order to ensure timely med-

ical treatment. Health monitoring alone may be preferred

where resources are limited.

The risk assessment approach presented here could be fur-

ther improved if an experimental probability distribution of the

estimated dose was available. However, such a probability dis-

tribution was not available even for the most common matrix

(soil). To obtain such a distribution, a large number (e.g., 30)

of different true doses must be spiked in the environmental

matrix of interest, and the sample must be processed through

an entire protocol. This time-consuming process has not yet

been reported.

Conclusion. Instrument and environmental limits of detec-

tion are necessary for QMRA when evaluating exposure to

human pathogens in a contaminated environment. Due to the

lack of pertinent data on the detection of B. anthracis, the

environmental limit of detection could not be represented as a

distribution. These distributions were necessary for estimating

the risk at the environmental limit of detection. Even so, it was

clear that environmental samples may be expected to have

broad distributions due to the many challenges in sample pro-

cessing that affect the limit of detection. More environmental

detection studies need to be conducted in order to produce

distributions similar to those of the instrument limit of detec-

tion. This will improve the risk assessment and improve the

applicability of the information in regard to survival and

cleanup goals, providing valuable information for ﬁrst re-

sponders.

ACKNOWLEDGMENTS

This research has been supported by the Center for Advancing

Microbial Risk Assessment, funded by the U.S. Environmental Pro-

tection Agency Science to Achieve Results (STAR) program, and U.S.

Department of Homeland Security University Programs grant

R83236201.

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