To the Editor: During the 2001 anthrax outbreak, we evaluated and validated a highly
sensitive and specific three-target (two plasmid and one chromosomally located target)
5´ nuclease assay (real-time polymerase chain reaction [PCR]) for detection and identification
of Bacillus anthracis. This PCR assay was successfully used to rapidly test hundreds
of suspect isolates as well as screen environmental samples for the presence of B.
anthracis throughout the 2001 anthrax outbreak. For the first time in an outbreak
setting, a PCR assay was used to detect B. anthracis directly from clinical specimens,
consequently becoming a part of the laboratory confirmation of anthrax. In this letter,
we describe the evaluation of this assay on a diverse panel of bacterial isolates
including isolates obtained throughout the outbreak. A supplement, which includes
data on the use of this assay on environmental and clinical specimens, is online (available
from http://www.cdc.gov/ncidod/EID/vol8no10/02-0393sup.htm).
Identification of B. anthracis has traditionally been determined by using phenotypic
differences between B. anthracis and the rest of the B. cereus group (i.e., lack of
motility and hemolysis, susceptibility to penicillin, typical colony morphology, and
susceptibility to lysis by gamma phage); however, these methods are slow and require
at least 24 h for completion. The recent bioterrorism-associated outbreak and the
ongoing threat emphasize the importance of rapid microbiologic diagnosis for the timely
and adequate implementation of control and preventative measures.
For B. anthracis, the main targets for development of such assays, primarily PCR-based,
have been and continue to be genes encoding its virulence factors: a tripartite exotoxin
and an antiphagocytic capsule (1–4). The toxin genes (pagA, lef, and cya) are encoded
on the 182-kb virulence plasmid, pXO1, while the genes required for capsule biosynthesis
(capB, capC, and capA) are encoded on the 96-kb virulence plasmid, pXO2 (5–7). These
plasmid-located virulence genes seem to be restricted to B. anthracis, giving the
plasmid-based assays a high degree of specificity (8). However, strains of B. anthracis
that lack these plasmids have been reported (4,9). Consequently, having an assay focus
on a specific chromosomal target for detection of avirulent and plasmid-cured B. anthracis,
as well as those that potentially could have been genetically engineered, is essential.
Chromosomal markers, such as vrrA and Ba813, have been used to characterize B. anthracis
(9–12) and to detect it in tissues of victims of the anthrax outbreak that occurred
in 1979 in Sverdlovsk, former Soviet Union (12), but these markers are not restricted
to B. anthracis. Recently, Qi et al. developed a fluorescence resonance energy transfer
PCR assay that targets the B. anthracis chromosomally located rpoB gene. This assay
appears to be the most specific described to date with only 1 of 175 non-B. anthracis
bacilli reported as positive (13).
Over the past several years, activities in the area of bioterrorism preparedness in
the United States have resulted in the establishment of an international Laboratory
Response Network (LRN), which was instrumental in the identification of the agent
used in the 2001 outbreak (14). One of the major initiatives of LRN has been development
and validation of rapid and specific assays for identification of B. anthracis and
other agents likely to be used in a bioterrorism event.
Primer and probe set BA1 targets a region of pX02, BA2 targets pX01, and BA3 targets
a region of the B. anthracis chromosome. Probes were labeled with 6-carboxy-fluorescein
phosphoramidite and 5-carboxy-tetramethyl-rhodamine.
LRN PCR assays using the BA1, BA2, and BA3 primer and probe sets were performed with
the LightCycler (Roche Diagnostics GmbH, Mannheim, Germany), Smart Cycler (Cepheid,
Sunnyvale, CA), or ABI Prism 7700 (Applied Biosystems, Foster City, CA) instruments.
The LightCycler Faststart DNA master hybridization probes kit (Roche Diagnostics GmbH)
reagents were used on all real-time platforms. Reactions comprised 1X reaction mix,
5 mM MgCl2, 500 nM each primer, and 100 nM probe in a reaction volume of 20 µL (LightCycler)
or 25 µL (Smart Cycler, ABI Prism 7700). Thermal cycler conditions consisted of an
initial 10-min hold at 95°C followed by 40–45 cycles of 10 s (LightCycler) or 15 s
(Smart Cycler, ABI Prism 7700) at 95°C and 30 s (LightCycler, Smart Cycler) or 60
s (ABI Prism 7700) at 60°C. Real-time data were collected during the 60°C extension
step of each cycle. Amplification of the human βbeta-actin gene was used as a real-time
PCR control when used on clinical samples to ensure negative results were not from
inhibition of the PCR reaction. This real-time PCR assay was considered positive when
all three targets were positive (Figure).
Figure
Real-time polymerase chain reaction graph of three B. anthracis markers and B-actin
control detected in a pleural fluid specimen from a patient with inhalational anthrax.
The horizontal line indicates a threshold value; the vertical lines indicate cross-threshold
values for each marker. BA1, primer and probe set targeting a region of pXO2; BA2,
primer and probe set targeting a region of pXO1; BA3, primer/probe set targeting a
region of B. anthracis chromosome.
A total of 542 isolates were tested. Eighty-one B. anthracis isolates were tested
to evaluate sensitivity of the real-time PCR approach (Table). Seventy-five were selected
to provide a test population representing diverse sources, genotypes, geographic origins,
and dates of isolation. The isolates included those collected from animals, humans,
and other sources (i.e., industrial sites associated with anthrax outbreaks); the
isolates span at least 58 years (1939–1997). Fifty-three of the isolates were previously
characterized by multiple-locus variable-number tandem repeat analysis (MLVA) (15)
and were included to ensure a representative range of the 89 described MLVA genotypes
to date. Six B. anthracis type and standard strains included: five pXO1 cured strains
(including the Pasteur strain) and one pXO2 cured strain (the veterinary vaccine strain
Sterne). The B. anthracis New Hampshire strain (16) was used as a positive control
for all real-time PCR assays. This isolate was originally cultured from a patient
with inhalational anthrax in New Hampshire in 1957. This real-time PCR is designed
to identify fully virulent (wild-type) B. anthracis, which will give positive results
in all three markers. However, naturally occurring isolates have been found lacking
either virulence plasmid, and a number of laboratory strains have been plasmid cured,
as well. PCR results for these strains will reflect the lack of one or both of their
plasmids.
Table
Origin, designations, and results of real-time polymerase chain reaction assay for
Bacillus
anthracis strainsa
No. positive/total
B. anthracis
No. analyzed
Temporal range and geographic origin
MLVA genotypes representeda
Ba1b
Ba2b
Ba3b
Human isolates
30
1943–1996 Africa, Asia, Australia, Europe, North America
3, 4, 22, 23, 28, 32, 34, 35, 36, 37, 41, 43, 44, 45, 50, 66, 68
30/30
30/30
30/30
Animal isolates
29
Africa, Asia, Australia, Europe, North America, South America
3, 10, 20, 26, 29, 30, 35, 38, 40, 45, 48, 49, 51, 55, 57, 78, 80, 81, 84, 85, 87,
89
29/29
29/29
29/29
Other isolates
16
1950–1993
Africa, Asia, Europe, N. America
13, 14, 21, 24, 47, 62, 69, 73, 77, 79, 82
16/16
16/16
16/16
Outbreak isolates
317
2001
U.S. outbreak
62
317/317
317/317
317/317
pXO1 cured
5
1956–1974
North America
5/5
0/5
5/5
pXO2 cured
1
Africa
0/1
1/1
1/1
aMLVA, multiple-locus variable-number tandem repeat analysis as described by Keim
et al. (15).
bBa1, Ba2, and Ba3 primer/probe sets as described in Materials and Methods.
A total of 317 B. anthracis isolates obtained during the bioterrorism-associated anthrax
outbreak from October to December 2001 were also analyzed by PCR. These included 27
isolates from clinical specimens, 4 from powders and 286 isolates from environmental
samples. MLVA was performed on 135 of these isolates; all were indistinguishable (17).
For evaluation of the assays’ specificity we tested 56 archived members of the Bacillus
genus: B. subtilis (9 strains, 5 clinical, 4 unknown), B. cereus (23 strains, 9 clinical,
14 environmental), B. thuringiensis (12 strains, 6 clinical, 3 insects, 3 unknown),
B. mycoides (1 strain, unknown), B. megaterium (10 strains, 7 clinical, 3 unknown),
and the environmental Bacillus spp. isolate, Ba813_11, which resulted in a previously
reported false-positive result in the B. anthracis-specific PCR assay targeting rpoB
(13). In addition, 88 isolates from environmental and clinical specimens, which were
confirmed not to be B. anthracis by standard microbiologic methods were tested. These
isolates were selected because of their lack of hemolysis and because they had a colony
morphology similar to B. anthracis on blood agar plates.
Before testing, all strains were stored at –70°C in brain heart infusion broth (BHIB,
Centers for Disease Control and Prevention [CDC], Atlanta, GA) or water containing
20% glycerol. Identification of all strains was confirmed by using standard microbiologic
procedures and the LRN testing algorithm (14,18). Colony-lysis DNA preparations were
used for all Bacillus spp. strains. Isolates were streaked onto trypticase soy agar
containing 5% sheep blood (Becton Dickinson Microbiology Systems, Cockeysville, MD)
and incubated overnight at 37°C. A single colony was transferred and dispersed into
0.22 mM centrifugal filter units (Millipore, Bedford, MA) containing 200 mL 10 mM
Tris-HCl (pH 8.0). The suspension was heated at 95°C for 20 min and then cooled to
room temperature. The filter units were then centrifuged at 6,000 x g in a microfuge
for 2 min and the filter discarded. The resulting lysate was stored at –20°C until
use.
The lower limit of detection of each assay was tested by using five B. anthracis strains:
Ames (2000031656), Pakistan-sheep (2000031648), French-bovine (2000031651), Sterne
(2000031075), and Pasteur (2000031759). DNA was extracted from vegetative cells by
first pre-treating cell pellets with lysozyme and lysostaphin and then using the MasterPure
DNA Purification kit (Epicentre, Madison, WI), following the manufacturer’s protocol
for cell samples. B. anthracis spores were quantitated microscopically and tested
directly in the real-time PCR assay without DNA extraction. Vegetative-cell DNA was
tested at concentrations ranging from 10 ng to 400 fg DNA per reaction. Spores were
tested at concentrations ranging from 100,000 spores to 1 spore per reaction. All
reactions were performed in duplicate on the LightCycler, Smart Cycler, and ABI Prism
7700 instruments.
All 75 wild-type (fully virulent) B. anthracis isolates tested were positive for all
three targets resulting in 100% sensitivity (95% confidence interval [CI] 95% to 100%).
Strains cured of pXO1 or pXO2 produced negative results for the loci specific to these
plasmids (Table). In addition, all 317 B. anthracis isolates from the 2001 outbreak
were also positive for all three PCR targets (Table).
None of the 56 archived non–B. anthracis isolates, representing five other Bacillus
species was positive for any of the three LRN PCR targets, including the Bacillus
spp. isolate, Ba813_11, resulting in 100% specificity (95% CI 94% to 100%). Results
were also negative for 88 clinical and environmental isolates, which were determined
by standard microbiologic methods not to be B. anthracis (specificity 100%, 95% CI
96% to 100%).
The limit of detection on the LightCycler, Smart Cycler, and ABI Prism 7700 instruments,
as determined by using DNA extracted from vegetative cells of the Sterne and Pasteur
reference strains, was 1 pg DNA (approximately 167 cells based on a 5.5 Mbp genome
size). Five to 10 spores could be detected on the ABI Prism 7700 instrument for the
Ames (2000031656), Pakistan-sheep (2000031648), French-bovine (2000031651), and Sterne
(2000031075) strains of B. anthracis.
The recent bioterrorism-associated anthrax outbreak demonstrated the need for sensitive,
specific, and rapid methods for diagnosis and confirmation of anthrax, both for identification
of suspect B. anthracis isolates and direct detection of B. anthracis DNA in clinical
specimens. When tested on >500 strains, representing B. anthracis and five other Bacillus
species, the LRN PCR exhibited 100% sensitivity and specificity.
To date, designing PCR assays for identification of B. anthracis has primarily focused
on genes located on the plasmids (1–4). Patra et al. used a PCR that targeted two
chromosomal loci, vrrA and Ba813, and found numerous environmental Bacillus isolates
other than B. anthracis that were positive for both Ba813 and vrrA (11). While assays
focusing on plasmid targets allow for a high level of specificity, a specific chromosomal
target for detection of avirulent and plasmid-cured B. anthracis strains is needed.
Thus, the LRN PCR includes a chromosomal target in addition to targets on each of
the two virulence plasmids, pXO1 and pXO2.
Closely related B. cereus and B. thuringiensis, notorious for generating false-positive
results using assays designed to be specific for B. anthracis (11,13), were consistently
negative in this real-time PCR assay. B. anthracis, B. cereus, and B. thuringiensis
are so closely related that their distinction as separate species is frequently questioned
based on DNA-DNA hybridization studies, multiple-locus enzyme electrophoresis, and
16S rRNA sequence similarity (19–21). We have selected non–B. anthracis isolates that
were primarily of clinical as opposed to environmental origin. B. cereus and B. thuringiensis
clinical isolates are even more closely related to B. anthracis than their environmental
counterparts (19,22), and they are more likely to cause false-positive results. We
also tested the Bacillus spp. isolate that caused the one false-positive result in
the Qi et al. report (13). Despite all of these challenges, all three targets of this
real-time PCR assay have demonstrated 100% specificity and sensitivity in identification
of B. anthracis when tested against our panel of Bacillus spp. strains and in identification
of 317 outbreak-associated B. anthracis isolates. This LRN PCR is currently the only
real-time PCR assay that detects both plasmid and chromosomal targets with 100% specificity
and sensitivity. In addition, real-time PCR assays using fluorescent probes provide
great sensitivity; this assay was able to detect 1 pg of purified DNA from vegetative
cells (equivalent to 167 cells) or directly detect 5–10 spores.
The high level of sensitivity and specificity of the LRN PCR assay can be attributed
to several factors. An extensive panel of DNA samples (non-Bacillus gram-positive
bacterial species, gram-negative bacterial species, and human, vertebrate, and invertebrate
DNA) were tested (data not shown). Having more than a single target decreases the
rate of both false-negative and false-positive results, as they are not dependent
on a single locus. The use of multiple targets also decreases the risk of false-positive
results from contamination because each target is amplified as a separate PCR reaction.
Finally, 5´ nuclease assays makes use of a fluorescent oligonucleotide probe, in addition
to the forward and reverse primers, that allows for a lower limit of detection compared
to conventional PCR, eliminates the need for post-PCR processing, and increases specificity
(23,24).
The LRN PCR was shown to be important for use on environmental and clinical specimens
during the 2001 bioterrorism-associated anthrax outbreak. A supplement covering the
use of this assay on these specimens can be seen online (available from http://www.cdc.gov/ncidod/EID/vol8no10/02-0393sup.htm).
The LRN PCR assay is widely available at over 200 laboratories in several countries
and all 50 states of the United States through the Laboratory Response Network. The
system is designed to be accessed through the State Department of Health.