Lab Q Clinical Laboratory No.

16 2014

12/14/15, 11:22 PM

Clinical Laboratory No. 16

2014
Microbiology

ISSN 2169-4702
print

Learning Objectives

Learning Objectives

History

On completion of this exercise, the participant should be able to

Discussion
References
CMLE Questions

discuss the epidemiology of brucellosis.

discuss why Brucella species are considered potential agents of bioterrorism.
list the characteristics of Brucella species and the laboratory tests to rule out such
organisms.
discuss potential exposure routes to Brucella species in the laboratory and
recommendations to prevent exposure and subsequent laboratory-associated
infection.

History
Case A
In June 2009, a 7-year-old girl was brought to her primary care physician with fever,
muscle pain, myalgia, and malaise. Blood cultures were drawn on June 29 and July 2
and sent to Laboratory A in Florida. Both blood cultures resulted in positive growth,
with the isolation of a slow-growing, gram-negative coccobacillus. The organism was
manipulated under biosafety level 2 (BSL-2) conditions on the open bench. The isolate
could not be identified and was sent to a more sophisticated diagnostic laboratory in
North Carolina (Laboratory B). Additional testing could not rule out Brucella species.
However, because Brucella was not initially on the laboratorys differential diagnosis,
the organism was again manipulated on the open bench. The isolate was sent to the
North Carolina State Laboratory of Public Health (NC SLPH), a Laboratory Response
Network (LRN) reference laboratory, where real-time polymerase chain reaction (RT-

Philip A. Lee,
MSc FIBMS
Lead Biological
Defense
Coordinator
Bureau of Public
Health Laboratories
Division of
Emergency
Preparedness and
Community
Support
Florida Department
of Health
Jacksonville,
Florida

Danielle Stanek,
DVM
Medical
Epidemiologist
Bureau of
Epidemiology
Division of Disease
Control and Health
Protection
Florida Department
of Health
Tallahassee, Florida

PCR) identified the organism as a Brucella species. Interviews with the patients family
revealed that the girl had recently helped dress a feral pig carcass. Because the
organism originated from a Florida resident, the NC SLPH forwarded the isolate to the
Florida Department of Health Bureau of Public Health Laboratories for speciation.
Case B
In November 2009, a 32-year-old woman experienced fever, malaise, and arthralgia.
On December 7, she sought treatment in the emergency department of her local
hospital, and blood cultures were drawn at that visit. Laboratory C isolated gramnegative coccobacilli, and on December 10 sent the organism to the NC SLPH, where
it was identified as a Brucella species by RT-PCR. The patient had no history of travel,
animal or animal product exposure, or consumption of raw dairy products.

BRUCELLA
Etiologic Agents
The International Committee on Systematics of Prokaryotes recognizes 10 species of
Brucella.1 Brucella abortus (cattle), Brucella melitensis (goats, sheep, and camels),
and Brucella suis (swine)are considered the most pathogenic to humans; Brucella
canis (dogs), Brucella pinnipedialis (seals), and Brucella ceti (dolphins and whales)
have been associated with human infections but are considered less pathogenic; and
Brucella ovis (sheep), Brucella neotomae (wood rats), and Brucella microti (voles)
have not been associated with human illness. Brucella inopinata is the newest member
of genus; it was first isolated from a breast implant wound in 2005,2 and a second

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strain was isolated from a lung biopsy specimen in 2009.3 The natural host for B
inopinata is not known.
Epidemiology of Brucellosis
Brucellosis is a zoonotic infection and is primarily a disease of cattle, swine, goats,
sheep, and dogs. Transmission to humans from animals occurs through direct contact
with infected secretions, including those from aborted fetuses or placentas, or
indirectly by ingestion of animal products and by inhalation of airborne agents.
Consumption of raw milk and cheese made from raw milk, especially sheep and goat
cheese, is the major source of infection in humans. Human brucellosis remains the
most common zoonotic disease worldwide, with more than 500,000 new cases
annually.4 Brucellosis presents an occupational disease for people who work with
livestock, particularly in endemic areas but also due to accidental inoculation of live
vaccines (such as B abortus strain 19 and B melitensis rev.1) in areas of the world
where brucellosis has been eradicated from domesticated herds. Human-to-human
transmission is very rare, but venereal and congenital infections have been reported.5

Quick Stop
Brucella is one of the top
10 causes of laboratoryassociated infection.
Brucella suis is endemic in
feral swine in the United
States.
Brucella species were the
first biological warfare
agents weaponized by the
United States.

Brucella suis is endemic in the feral swine population in the United States. Feral swine
are found in numerous US states, with especially high concentrations in Florida,
California, Texas, and Hawaii.6 This presents a hazard for hunters who come in
contact with blood and tissues from infected pigs. In recent years, B abortus and B
melitensis associated with international travel to developing countries has also
increased, owing to exposure to unpasteurized milk products in those countries.
Case Definition and Classification
The Council of State and Territorial Epidemiologists (CSTE) case definition for human
brucellosis is an illness characterized by acute or insidious onset of fever and 1 or
more of the following: night sweats, arthralgia, headache, fatigue, anorexia, myalgia,
weight loss, arthritis/spondylitis, meningitis, or focal organ involvement (endocarditis,
orchitis/epididymitis, hepatomegaly, splenomegaly).7 Neurologic symptoms may occur
acutely in up to 5% of cases. Neurobrucellosis is a rare cause of spastic paraparesis.8
The mortality rate is 2% or less, and death is usually due to endocarditis and other
cardiovascular complications. Relapse is relatively common, and in low-incidence
regions, the nonspecific symptoms of brucellosis often result in initial misdiagnosis.
A probable case is classified as a clinically compatible illness with at least 1 of the
following:
Epidemiologically linked to a confirmed human or animal brucellosis case
Presumptive laboratory evidence, but without definitive laboratory evidence, of
Brucella infection
A confirmed case is classified as a clinically compatible illness with definitive
laboratory evidence of Brucella infection.7
Alternate names for brucellosis include Bang disease, Gibraltar fever, Malta fever,
Maltese fever, Mediterranean fever, rock fever, and undulant fever.
Bioterrorism Aspects
Designated Agent US, B suis was the first biological warfare agent weaponized by the
United States at Pine Bluff Arsenal, Arkansas, in 1954.9 Subsequently; B abortus
(Agent AB) and B melitensis (Agent AM) were similarly weaponized.
As such, the Centers for Disease Control and Prevention (CDC) classifies Brucella
species as Category B agents of bioterrorism. These agents are moderately easy to
disseminate, cause moderate morbidity rates and low mortality rates, and require
specific enhancements of CDCs disease surveillance and diagnostic capacity through
the LRN.10 Owing to their natural properties as well as their potential to be used as
biological weapons, B abortus, B melitensis, and B suis are categorized as select
agents under the Public Health Security and Bioterrorism Preparedness and Response
Act of 2002. When an agent is identified from a diagnostic specimen, the CDC Division
of Select Agents and Toxins and/or the US Department of Agriculture Animal and Plant
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Health Inspection Service (APHIS) must be notified and APHIS/CDC Form 4 completed.
If there was a possibility of an exposure, APHIS/CDC Form 3 must also be
completed.11 In addition, all cases of suspected and confirmed brucellosis should be
reported to the state health department and the CDCs National Notifiable Diseases
Surveillance System.
As with other agents, additional state and federal notification systems exist if Brucella
species is identified in association with a potential bioterrorism event. The Federal
Bureau of Investigation (FBI) Weapons of Mass Destruction Directorate would be
notified as the lead federal law enforcement agency tasked to investigate the criminal
aspect of such an event.
Laboratory Response Network
The LRN was established in 1999 by the CDC, FBI, and the Association of Public
Health Laboratories. It is an integrated network of more than 150 state and local
public health, food safety, veterinary, federal, military, and international reference
laboratories that can respond quickly to needs for rapid testing, timely notification,
and secure messaging of results associated with acts of biological or chemical
terrorism and other high-priority public health emergencies.12
The estimated 25,000 sentinel clinical laboratories in the United States play a key role
in the early detection of bioterrorism agents, by either ruling out such agents or
referring specimens and isolates to LRN reference laboratories for confirmatory
testing.
Laboratory Diagnosis
Laboratory criteria for the diagnosis of clinical brucellosis in humans and animals is as
follows.7
Presumptive laboratory diagnosis:
Brucella total antibody titer of 160 or greater by standard tube agglutination test
(SAT) or Brucella microagglutination test (BMAT) in 1 or more serum specimens
obtained after onset of symptoms;
Detection of Brucella DNA in a clinical specimen by PCR assay. Definitive laboratory
diagnosis:
Culture and identification of Brucella species from clinical specimens;
Evidence of a 4-fold or greater rise in Brucella antibody titer between acute-phase
and convalescent-phase serum specimens obtained 2 weeks or more apart.
Commercially available Brucella immunoglobulin M (IgM) and IgG enzyme-linked
immunosorbent assay (ELISA) serologic tests have occasionally resulted in falsepositive or false-negative results in regions of low endemicity.13 As such, the SAT and
BMAT assays are considered the criterion standard for Brucella abortus, B
melitensis,and B suis serology and are performed by the CDC and some LRN reference
laboratories.
Brucella species are most often isolated from whole blood and bone marrow. However,
the organism may also be isolated from spleen, liver, and a wide variety of body
tissues such as joint fluid, cerebrospinal fluid, semen, pulmonary excretions, placenta,
and occasionally urine.
Diagnostic laboratories should follow the American Society for Microbiology (ASM)
Sentinel Level Clinical Microbiology Laboratory Guidelines for Suspected Agents of
Bioterrorism and Emerging Infectious Diseases.14 Brucella species cannot be ruled out
if an isolate exhibits the following characteristics: dysgonic (slow-growing) aerobic
growth; nonmotile gram-negative coccobacillus, 0.5-0.7 m 0.6-1.5 m; 48 to 72
hours to see individual colonies, particularly from clinical specimens; grows on most
laboratory media, but no growth on MacConkey or eosin methylene blue (EMB) agar
(may be pinpoint at 7 days); small, convex, glistening colonies with an entire edge; is
nonhemolytic and nonpigmented (Image); catalase positive; does not satellite around
Staphylococcus aureus; oxidase positive, and urease positive. The isolate should be
referred directly to an LRN reference laboratory for rapid RT-PCR analysis and species
confirmation testing. Isolates should not be sent to a more sophisticated sentinel
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laboratory. Brucella species are LRN priority organisms, and all LRN reference
laboratories have the capability of rapidly identifying Brucella to the genus level by
RT-PCR within a few hours of receiving an isolate. Speciation is performed by more
conventional microbiologic analyses and takes several days, owing to the slow growth
of the organism.
Other urea-positive, oxidase-positive, gram-negative coccobacilli likely to be
encountered by clinical laboratories include Bordetella bronchiseptica, Bordetella hinzii,
Cupriavidus pauculus, Haemophilus influenzae, Oligella ureolytica, Paracoccus yeei,
Psychrobacter immobilis, Psychrobacter phenylpyruvicus, and oxidase-producing
strains of Aggregatibacter actinomycetemcomitans. Brucellae must be differentiated
from these similar organisms. In addition, Brucella species are often misidentified
because they do not always decolorize on Gram stain and can appear as gram-positive
or gram-variable. They have been reported as gram-positive cocci, coryneform bacilli,
and Micrococcus. Historically, this retention of gram-positivity was documented by
British Army physician David Bruce, for whom the genus is named. In 1887, Bruce
isolated the organism from the spleens of 5 patients with fatal cases on the island of
Malta in the Mediterranean and named the new species Micrococcus melitensis. 15
The use of automated identification systems for Brucella species is discouraged.
Preparation of a standard concentration of the organism in a liquid suspension for use
in these systems presents the potential to generate a widespread aerosol of infectious
organism. Also, many automated systems use phenotypic methods to produce
biochemical profiles of the unknown organism that are then matched to known
organisms in their database. These systems typically produce high-confidence
identifications for rapidly growing organisms. However, slow-growing organisms like
Brucella tend to produce low-confidence results or are misidentified because
insufficient enzymes are produced during the limited incubation times. In addition,
databases for infrequently encountered organisms are often built on very few isolates,
which can exacerbate the inaccuracy of such automated identification systems. There
are several reports in the literature where Brucella species have been misidentified as
Ochrobactrum anthropi using automated systems.16-18
Laboratory Biosafety
Owing to the ease of aerosolization and the highly infectious nature of the organism
(infective dose: 10-100 organisms), brucellosis has been one of the 10 most
commonly reported laboratory-associated infections.19 BSL-2 practices, containment
equipment, and facilities are recommended for working with routine clinical specimens
and provide adequate protection, owing to the low bacterial loads found in human
specimens. Once isolated in culture and propagated in large numbers of organisms,
biosafety level 3 (BSL-3) practices, containment equipment, and facilities are
recommended for all manipulation of pathogenic Brucella species. Sniffing bacteriologic
cultures and manipulating cultures on open bench tops under BSL-2 conditions have
resulted in potential exposures requiring a laboratory risk assessment and medical
intervention.
According to the CDC, the laboratory risk assessment for exposure to Brucella species
is categorized into 3 risk groups; high, low, and none. The high-risk category is
further subdivided.20
High-risk individuals: Those who manipulated the organism on the open bench or
worked with the organism in a Class II biological safety cabinet (BSC), but did not
use

BSL-3 practices;

High-risk close proximity: All individuals within a 5-foot radius of work with Brucella
species outside of a Class II BSC on an open bench, but work did not involve
widespread aerosol-generating procedures;
High-risk laboratory room: All personnel present in the room when Brucella was
handled on the open bench when widespread aerosol-generating procedures were
performed;
Low-risk laboratory room: Personnel present in the laboratory at the time of
manipulation of Brucella on an open bench, but who do not have high-risk
exposures
as defined above;

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No risk: Handling and testing of Brucella in a Class II BSC using BSL-3 precautions.
Postexposure prophylaxis (PEP) is recommended for all personnel meeting the criteria
for a high-risk exposure. PEP may be offered to personnel with a low-risk exposure,
but is not required for those in the no-risk category. PEP consists of a minimum of 21
days of doxycycline (100 mg orally, twice daily) and rifampin (600 mg, once daily).
Where doxycycline is contraindicated, trimethoprim-sulfamethoxazole (160 mg/800 mg
orally, twice daily) may be prescribed for at least 21 days. In addition to PEP,
recommendations indicate that all exposed personnel should undergo serologic
monitoring. Prior to November 2012, serologic analyses were performed at 0, 2, 4, 6,
and 24 weeks after exposure.21
Laboratory Exposures to Brucella species
During a 17-month period beginning January 2009, 13 cases of brucellosis were
identified in Florida, including case A discussed here, which resulted in the laboratoryassociated infection in North Carolina. Of these, the Florida Department of Health
investigated 11 cases that involved laboratory exposures to Brucella culture isolates.
The cases included 1 B abortus in a traveler to El Salvador; 3 B melitensis in travelers
to Mexico, Egypt, and Syria; and 7 B suis, 6 of which were related to feral swine
hunting.
An isolate that cannot be identified locally is often sent to a more sophisticated
clinical laboratory for further analysis. Thus, the 11 cases involved 18 clinical
laboratories before being referred to an LRN reference laboratory for confirmatory
testing. Because the organism had not yet been identified, inadequate biosafety
practices for Brucella species in 15 of the 18 laboratories resulted in 71 high-risk
exposures and 74 low-risk exposures. All 145 personnel were offered PEP and active
serologic monitoring, resulting in more than 500 serum specimens undergoing BMAT
serology. Passive medical evaluation for fever and clinical symptoms was also
established.
Causes for the exposures were various. In one case, there was a clinical suspicion of
brucellosis, but the information was not communicated to the laboratory. Another case
resulted in 4 additional high-risk exposures when information was not communicated
internally within the laboratory once Brucella species was suspected. IgM and IgG
ELISA serologic tests are often performed by different clinical laboratories than those
performing bacterial isolation on specimens from the same patient. Occasionally, a
positive result on a serologic test result was available before the organism was
isolated in culture, but this essential information was not communicated in a timely
manner to the laboratory performing the culture, resulting in preventable exposures.
Once isolated in culture, the rarely encountered organism is not always on the
laboratory differential diagnosis: other urea-positive, oxidase-positive gram-negative
coccobacilli, listed above, are more commonly encountered in the clinical microbiology
laboratory. Several exposures resulted from the failure of the organism to decolorize
on Gram stain with initial reports of Micrococcus isolation, and failure of the oxidase
test (dry format) led to 1 exposure. Misidentification of Brucella species as
Ochrobactrum anthropi using automated systems occurred in at least 2 instances in
Florida, leading to further exposures. Finally, sniffing cultures resulted in high-risk
exposures in 2 laboratories. This practice is never recommended, whether the isolate
identity is believed to be known or not.
CDC Recommendations
The following are the current CDC recommendations for safe laboratory practices when
working with Brucella species to prevent exposures and laboratory-associated
infections21:
When brucellosis is suspected, clinicians should note "suspect [or rule out]
brucellosis" on the laboratory submission form.
Review laboratory containment methods and microbiologic procedures to ensure
compliance with recommendations of the 5th edition of the CDC's Biosafety in
Microbiological and Biomedical Laboratories (BMBL).19
Use primary barriers: safety centrifuge cups, personal protective equipment, and
Class II or higher BSCs for procedures with a high likelihood of producing droplet

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splashes or aerosols.
Use secondary barriers: restrict access to the laboratory when work is being
performed.
Maintain the integrity of the laboratory's air-handling system by keeping external
doors and windows closed.
Perform all procedures on unidentified isolates carefully to minimize the creation of
splashes or aerosols.
Prohibit sniffing of opened culture plates to assist in the identification of isolates.
Manipulate all isolates of small, slow-growing, gram-negative or gram-variable rods
within a BSC.
Because multiple laboratory-associated infections with Brucella species have occurred
in recent years, modifications to the recommendations for safe laboratory practices
have been suggested.22 Modifications include more active medical evaluation for fever
and clinical symptoms in exposed personnel in addition to a change in timeline for
postexposure serologic monitoring from 0, 2, 4, 6, and 24 weeks to 0, 6, 12, 18, and
24 weeks postexposure.20
Case Conclusions
Case A
The final identification of the organism from this patient was B suis, which matched
the epidemiologic data. Manipulations of this highly infectious organism on the open
bench resulted in 5 high-risk and 8 low-risk exposures at Laboratory A and 5 high-risk
and 1 low-risk exposure at Laboratory B. All 13 exposed personnel at Laboratory A
and the 6 exposed at Laboratory B were offered PEP and medical evaluation through
serologic monitoring. All serologic analyses were negative at 6 weeks postexposure.
Case B
This patient was a microbiologist who worked at Laboratory B where she had been
exposed to B suis between July 13 and July 18, 2009. She had started PEP on July 27
and completed the 21-day treatment. Because the serologic results were negative at 6
weeks postexposure, she assumed the PEP was successful and did not inform the
emergency room physician of her exposure to Brucella 5 months previously. Therefore,
brucellosis was not clinically suspected, and when it was isolated, it resulted in 11
high-risk exposures in Laboratory C. Subsequently, her 24-week serologic result was
positive.
Summary
With international travel and feral swine expanding in both numbers and geographic
range, there is a potential for encountering Brucella more often in the laboratory.
Introduction of the stringent reporting requirements of select agent regulations has
heightened awareness of possible Brucella exposures and required medical evaluation
of laboratory personnel. Subsequent to these findings, the Florida Department of
Health increased outreach activities to educate laboratory personnel about the
potential risks, to increase awareness in physicians and educate them in the
importance of communicating a suspicion of brucellosis to the laboratory, and finally
to educate hunters of feral swine on methods of protecting themselves and the
importance of reporting their hunting status to primary care providers if they become
ill. In 2011 and 2012, the Florida Department of Health responded to significantly
fewer laboratory exposures. The ultimate goal is to implement effective safety policies
and practices designed to prevent Brucella cases, exposures, and laboratoryassociated infections.

KEY TO IMAGES

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Image. Brucella melitensis colonies on 5% sheep blood agar, 72-hour incubation. CDC
Public Health Image Library, accession # 1902. Image courtesy of Larry Stauffer,
Oregon State Public Health Laboratory.

References
1. International Committee on Systematics of Prokaryotes, Subcommittee on the
Taxonomy of Brucella. Correct names of taxa within the genus Brucella with
details of type and reference strains. International Committee on Systematics of
Prokaryotes website. http://www.the-icsp.org/taxa/Brucellalist.htm
(http://www.the-icsp.org/taxa/Brucellalist.htm) . Published July 2010. Accessed April
14, 2013.
2. Scholz HC, Nckler K, Gllner C, et al. Brucella inopinata sp. nov., isolated from a
breast implant infection. Int J Syst Evol Microbiol. 2010;60:801-808.
3. Tiller RV, Gee JE, Lonsway DR, et al. Identification of an unusual Brucella strain
(BO2) from a lung biopsy in a 52-year-old patient with chronic destructive
pneumonia. BMC Microbiology. 2010; 10:23-33.
4. Pappas G, Papadimitriou P, Akritidis N, et al. The new global map of human
brucellosis. Lancet Infect Dis. 2006;6:91-99.
5. WHO. Brucellosis in humans and animals. Geneva, Switzerland: World Health
Organization; 2006.
6. Southeastern Cooperative Wildlife Disease Study. National feral swine mapping
system. University of Georgia College of Veterinary Medicine website.
http://128.192.20.53/nfsms/ (http://128.192.20.53/nfsms/) . Published November
2012. Accessed April 14, 2013.
7. Centers for Disease Control and Prevention, Council of State and Territorial
Epidemiologists. 2012 Nationally notifiable diseases and conditions and current
case definitions. CDC website.
http://wwwn.cdc.gov/nndss/document/2012_Case%20Definitions.pdf
(http://www.cdc.gov/nndss/document/2012_Case%20Definitions.pdf) . Published 2012.
Accessed April 14, 2013.
8. Ahmed R, Patil BS. Neurobrucellosis: a rare cause for spastic paraparesis. Braz J
Infect Dis. 2009;13:245.
9. United States Army Medical Research Institute of Infectious Diseases. Medical
Management of Biological Casualties Handbook. 7th ed. Washington, DC:
Government Printing Office; 2011.
10. Centers for Disease Control and Prevention Emergency Preparedness and
Response. Bioterrorism Agents/Diseases by Category. CDC website.
http://emergency.cdc.gov/agent/agentlist-category.asp
(http://emergency.cdc.gov/agent/agentlist-category.asp) . Accessed December 10,
2013.
11. Federal Register. 42 CFR Part 73: Possession, Use, and Transfer of Select Agents
and Toxins. National Select Agent Registry website.
(http://www.selectagents.gov/Regulations.html)

http://www.selectagents.gov/Regulations.html
(http://www.selectagents.gov/Regulations.html) . Published April 2, 2013. Accessed
April 8, 2013.
12. Centers for Disease Control and Prevention Emergency Preparedness and
Response. Laboratory Response Network. CDC website.
http://emergency.cdc.gov/lrn/ (http://emergency.cdc.gov/lrn/) . Published July 2,
2012. Accessed March 12, 2013.
13. Pragle A, Blackmore C, Clark TA, et al. Public health consequences of a falsepositive laboratory test result for BrucellaFlorida, Georgia, and Michigan, 2005.
MMWR Morb Mortal Wkly Rep.2008;57:603-605.
14. American Society for Microbiology. Sentinel level clinical laboratory guidelines for
suspected agents of bioterrorism and emerging infectious diseases: Brucella
species. ASM website. http://www.asm.org/index.php/guidelines/sentinelguidelines (http://www.asm.org/index.php/guidelines/sentinel-guidelines) . Published
March 2013. Accessed April 4, 2013.
15. Moreno E, Moriyn I. Brucella melitensis: a nasty bug with hidden credentials for
virulence. Proc Natl Acad Sci. 2002;99:1-3.
16. Horvat RT, El Atrouni W, Hammoud K, et al. Ribosomal RNA sequence analysis of
Brucella infection misidentified as Ochrobactrum anthropi infection. J Clin
Microbiol. 2011;49:1165-1168.
17. Yang J, Ren X, Chu M, et al. Mistaken identity of Brucella infection. J Clin
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Microbiol. 2013;51:2011.
18. Carrington M, Choe U, Ubillos S, et al. Fatal case of brucellosis misdiagnosed in
early stages of Brucella suis infection in a 46-year-old patient with Marfan
syndrome. J Clin Microbiol. 2012;50:2173-2175.
19. US Department of Health and Human Services; Centers for Disease Control and
Prevention; National Institutes of Health. Biosafety in Microbiological and
Biomedical Laboratories (BMBL). 5th ed. Washington, DC: Government Printing
Office; 2009.
20. Centers for Disease Control and Prevention. Brucellosis: assessing laboratory risk
level and PEP. CDC website. http://www.cdc.gov/brucellosis/laboratories/risklevel.html (http://www.cdc.gov/brucellosis/laboratories/risk-level.html) . Published
November 2012. Accessed April 11, 2013.
21. Griffith J, Sullivan M, Howell J. Laboratory-acquired brucellosisIndiana and
Minnesota, 2006. MMWR Morb Mortal Wkly Rept. 2008;57:39-42.
22. Traxler RM, Guerra MA, Morrow MG, et al. Review of brucellosis cases from
laboratory exposures in the United States in 2008 to 2011 and improved
strategies for disease prevention. J Clin Microbiol. 2013;51:3132-3136.

CMLE Questions
If you would like to receive credit for this exercise, close this window to return to the
Educational Activity page and proceed to the Exercise Assessment.
1. Which of the following statements about the epidemiology of brucellosis is true?
a. Brucellosis is primarily a human infection.
b. Consumption of raw milk and cheese made from raw milk is the major source of
infection in humans.
c. Brucella melitensis presents a hazard to feral swine hunters in the United States.
d. Brucellosis is a rare zoonotic disease worldwide.
2. Which of the following statements is true regarding Brucella species as potential
agents of bioterrorism?
a. Brucella species cause high human morbidity and mortality rates.
b. The Centers for Disease Control and Prevention (CDC) classifies Brucella species
as Category A agents of bioterrorism.
c. Brucella detection requires specific enhancements of CDCs disease surveillance
and diagnostic capacity through the Laboratory Response Network (LRN).
d. Brucella canis is categorized as a select agent under the Public Health Security
and Bioterrorism Preparedness and Response Act of 2002.
3. Which of the following sets of laboratory results would require the organism to be
sent to an LRN reference laboratory for Brucella confirmation testing?
a. Dysgonic growth on agar; nonhemolytic; nonmotile gram-negative coccobacillus;
no growth on MacConkey at 48 h; catalase positive; does not satellite around
Staphylococcus aureus; oxidase positive; urease negative.
b. Dysgonic growth on agar; nonhemolytic; nonmotile gram-negative coccobacillus;
no growth on MacConkey at 48 h; catalase positive; satellites around S aureus;
oxidase positive; urease positive.
c. Dysgonic growth on agar; nonhemolytic; motile gram-negative coccobacillus;
growth on MacConkey at 48 h; catalase positive; does not satellite around S
aureus; oxidase positive; urease positive.
d. Dysgonic growth on agar; nonhemolytic; nonmotile gram-negative coccobacillus;
no growth on MacConkey at 48 h; catalase positive; does not satellite around S
aureus; oxidase positive; urease positive.
4. Which of the following is considered low risk for exposure to Brucella species in the
laboratory?
a. Handling and testing of Brucella in a Class II biological safety cabinet (BSC)
using biosafety level (BSL)-3 precautions.
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b. Individuals within a 5-foot radius of work with Brucella species outside of a Class
II BSC on an open bench, but work did not involve widespread aerosol-generating
procedures.
c. Personnel present in the laboratory at the time of manipulation of Brucella on an
open bench greater than a 5-foot radius, not involving widespread aerosolgenerating procedures.
d. Personnel who manipulated the organism on the open bench.
5. Which of the following is included in the current CDC recommendations for safe
laboratory practices when working with Brucella species to prevent exposures and
laboratory-associated infections?
a. Use automated identification systems to rapidly confirm Brucella species.
b. Review laboratory containment methods and microbiological procedures to ensure
compliance with recommendations of the 5th edition of Biosafety in Microbiological
and Biomedical Laboratories (BMBL).
c. Practice sniffing of opened culture plates to assist in the identification of isolates.
d. Manipulate all isolates of large, rapidly growing gram-negative or gram-variable
rods within a BSC.

Copyright 2014 ASCP

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