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A review of sentinel laboratory performance: identification and notification of bioterrorism agents.

In 2001, an anthrax incident in the United States prompted extensive review of how clinical and public health laboratories should respond to bioterrorism. The first 2001 anthrax case was fortuitously identified in Florida as part of an investigation of altered mental status and severe respiratory disease in a patient who handled mail for a publishing company. (1) Subsequently, other cases were identified in New York, Connecticut, and Washington, District of Columbia that were typically associated with received mail or mailroom occupations. Recognizing the need for a network of laboratories capable of detecting, confirming, and reporting potential bioterrorism agents, the Centers for Disease Control and Prevention (CDC), the Federal Bureau of Investigation (FBI), and the Association for Public Health Laboratories (APHL) developed the Laboratory Response Network (LRN) in 1999. (2) Since the network was put in place in 1999, numerous sentinel laboratories across the country have been trained by their LRN reference laboratories and are familiar with biothreat agents, such as Bacillus anthracis.

The mission of the LRN is to "... maintain an integrated national and international network of laboratories that are fully equipped to respond quickly to acts of chemical or biological terrorism, emerging infectious diseases, and other public health threats and emergencies." (3) The LRN is typically presented as a pyramid. At the base of a diagrammatic pyramid are the sentinel laboratories (Figure 1). These are the clinical laboratories commonly found in or associated with hospitals or larger reference laboratories, where the primary identification of an infectious agent occurs. There are thousands of such laboratories in the United States. The American Society for Microbiology (ASM) provides specific guidelines for sentinel laboratories in support of their important role in the LRN laboratory system. (4) An advanced sentinel laboratory is one "... capable of analyzing or referring specimens or samples that may contain microbiology agents or biological toxins." (4,5) Basic sentinel laboratories should be CLlA (Clinical Laboratory Improvement Amendments of 1988) certified for nonwaived testing and should have policies and procedures for referral of diagnostic specimens. It is recommended that advanced sentinel laboratories additionally have a Class II or higher certified biologic safety cabinet and comply with biosafety level II (BSL-2) practices.


Reference laboratories in the schematic in Figure 1 are largely represented by state and local public health laboratories. There are approximately 160 reference laboratories whose role is to confirm or rule out suspected bioterrorism organisms, thereby producing high-confidence test results for threat analysis and for intervention by public health and public safety authorities. At the apex of the pyramid are the national laboratories that definitively characterize samples and microbial isolates. The CDC and the US Army Medical Research Institute for Infectious Diseases (USAMRIID) laboratories also have special containment with biosafety level IV (BSL-4) facilities.

With the introduction of the LRN, it became imperative to educate and train sentinel laboratory personnel in the identification and reporting mechanisms for potential bioterrorism agents. In one of several mechanisms for training sentinel laboratorians, the College of American Pathologists (CAP), in collaboration with the CDC and APHL, created in 2007 a bioterrorism response educational exercise and began sending attenuated or vaccine strains of bioterrorism agents to achieve the following goals.

1. To provide LRN sentinel laboratories with a realistic bioterrorism agent challenge exercise.

2. To provide an educational exercise that would test most aspects of a laboratory bioterrorism response, to include the following:

a. Ruling out and referral of potential bioterrorism agents (both surrogates and attenuated "real" organisms) by using appropriate LRN Sentinel Laboratory Guidelines.

b. Notification by the participating LRN sentinel laboratory to the appropriate local LRN reference laboratory of potential bioterrorism organisms.

c. Packaging and shipping of organisms to the appropriate local LRN reference laboratory (some laboratories may be asked by their LRN reference laboratory to participate in exercises to determine the effectiveness of packaging and shipping specimens in their state).

d. Knowledge of appropriate laboratory protocols that address the safe handling of highly pathogenic organisms.

3. To provide information to state and local public health LRN reference laboratories about gaps in the LRN sentinel laboratory system or gaps in communication between the reference and sentinel laboratories in each jurisdiction, so that the LRN reference laboratories set appropriate focus on developing and offering laboratory bioterrorism training and education to address these gaps.

The CAP bioterrorism exercises discussed here are one of the first national programs to examine the capabilities of sentinel laboratories for ruling in and ruling out a bioterrorism agent and for examining the LRN reporting procedures for the participating laboratories. We have summarized the experience with the bioterrorism exercises, as provided by CAP for 2007 and 2008.



Participant laboratories selected the Laboratory Preparedness Survey (LPS) A and B mailings (2007) and/or the Laboratory Preparedness Exercise (LPX) A and B mailings (2008), as offered annually by CAP through the CAP Surveys catalog. Subscribers were explicitly informed that bioterrorism agents might be part of each challenge and that sterilization and appropriate disposal of the provided agents was required after performance of the exercise. Also, participating laboratories were required to "opt in" to the LPX exercise by submitting a signed affidavit stating that their laboratory was equipped with a certified Class II biosafety cabinet and indicating that they would comply with BSL-2 practices (equivalent to an LRN advanced sentinel laboratory). Participants were cautioned to use appropriate biosafety practices for any procedures requiring subculture and other manipulations of specimens during the exercise. Participants were encouraged to discuss with their respective LRN reference laboratories whether actual specimen submission was required for the challenge specimens. Participating laboratories were also advised to have in place appropriately trained personnel and supplies for packaging and shipment of biologic materials.


The organisms used in LPS (A and B) and LPX (A and B) are shown in Table 1. Each of the 4 challenges contained 5 organisms. Category A and category B bioterrorism agents were included in each mailing. (6) Category A agents pose the greatest threat because they are easily disseminated and highly infectious. Category B agents are moderately easy to disseminate and have moderate morbidity and mortality. Other organisms represented mimics of select agents or standard clinical isolates that might be received in the midst of a bioterrorism event. Organisms were manufactured as lyophilized swabs and provided in a foil pouch, with instructions for reconstitution using the provided rehydration fluid. Susceptibility testing on challenge organisms was performed by the CDC Division of Healthcare Quality Promotion, and a certificate of analysis was provided to the CAP for each organism and with each challenge. The certificate of analysis included purity and growth characteristics, gram-stain morphology, reaction results for motility, oxidase, catalase, urea, indole, growth at 25[degrees]C, and susceptibility-testing results.

Exercise Design

Each challenge isolate was received by the testing laboratory with instructions for setting up culture from the lyophilized swab. Laboratories treated the specimens as if they were patient specimens (unknowns) in terms of identification procedures and posting of preliminary and final results. Laboratories were instructed to follow the ASM Sentinel Level Clinical Microbiology Laboratory Guidelines, which specify more limited identification, as recommended by their LRN reference laboratory or public health authorities if a bioterrorism agent is suspected in the course of the identification process. Instructions for the appropriate timing of notification were provided in 2008 (as soon as rapid biochemical and morphologic findings raised suspicion) compared to 2007. When the isolate was identified to a point appropriate for the given laboratory, a set of questions was provided regarding the identification procedures performed on the isolate. The isolate culture and biochemical characteristics queried are listed in Table 2.

Identification options ranged from full identification of the organism through a series of acceptable options, including genus level identification (unable to rule out); genus level identification (not otherwise specified); gram negative/positive (morphology, ie, bacilli), aerobic/anaerobic, "refer to rule out"; gram-negative/ positive (morphology, ie, bacilli), not otherwise specified. The identification options in 2008 were changed to full identification; (bioterrorism agent) refer to rule out; gram-negative/positive (morphology), refer to rule out (bioterrorism agent); and nonbioterrorism agent (full identification not required).

Options for notification or reporting of the results were as follows:

* Contact the appropriate local LRN reference laboratory and follow their instructions.

* Call the CDC.

* Refer the isolate to your normal commercial reference laboratory.

* Refer the isolate to the CDC.

* No further action should be taken.

* Other.

Also collected was the time interval between specimen processing and notification to an appropriate LRN reference laboratory about an identified or suspected bioterrorism agent.


LPS-A 2007

This mailing included Bacillus anthracis (Sterne strain), Bacillus megaterium, Burkholderia thailandensis, Yersinia pestis, and Klebsiella pneumoniae. B anthracis and B megaterium were sent as simulated blood cultures, with B megaterium presented as a mimic for B anthracis. Burkholderia thailandensis was presented as a mimic for Burkholderia pseudomallei and Burkholderia mallei, which are listed as select agents.

More than 90% of laboratories provided an acceptable response for B anthracis (1109 of 1231 participants) (Tables 3 and 4 [summary]). Only 5.4% fully identified the B anthracis isolate; however, large numbers of responders indicated that it was "Bacillus sp, unable to rule out B anthracis" (Table 3).

Bacillus anthracis (wild-type strains) are large, encapsulated, aerobic, gram-positive, catalase-positive, nonmotile bacilli that grow well on sheep blood agar, chocolate agar, and other gram-positive selective media, but not on MacConkey agar. This bacillus is usually detected in blood culture systems within 6 to 24 hours, while spores are not visible in primary blood culture bottles. Colonies are nonhemolytic on blood-containing media. The bacilli may form oval, central to subterminal spores, which do not swell the outlines of the bacillus when cultured aerobically without carbon dioxide. Also, B anthracis is positive for nitrates and negative for acid production from glycerol, inulin, mannitol, and salicin. (7)

Bacillus megaterium may be slightly [beta]-hemolytic and is usually motile. Also, it is negative for nitrates and positive for acid production for glycerol, inulin, mannitol, and salicin. The key tests for distinguishing B megaterium from B anthracis are motility and hemolysis. In this mailing, there was variation in the reporting of hemolysis production for B megaterium, perhaps related to this specific isolate. More disturbing, 45.0% (534 of 1188 participants) reported B megaterium as nonmotile and 17.7% (211 of 1188 participants) did not perform motility testing on this organism. As indicated in the ASM guidelines for sentinel laboratories in the presumptive identification of bioterrorism isolates, motility is a key test for determining whether a large, aerobic, gram-positive bacillus may potentially be B anthracis. However, it may be difficult to evaluate this feature accurately with the strain used in these trials. (4)

Burkholderia thailandensis (simulated wound isolate) was provided as a mimic for Burkholderia pseudomallei and Burkholderia mallei. Of 1243 participants in the identification exercise for this challenge, 347 (28.1%) identified the organism as B pseudomallei instead of B thailandensis (data not shown). Some participants (484, 39.2%) were unable to rule out B pseudomallei and indicated that they would call and/or refer to an appropriate LRN reference laboratory. The remaining participants identified this challenge as either B thailandensis or a gram-negative bacillus with appropriate chemical and culture characteristics (data not shown). Burkholderia mallei is the agent responsible for "glanders," a disease generally affecting livestock. Burkholderia pseudomallei produces diverse symptoms referred to as melioidosis, ranging from skin ulcers to septic shock. (8) Burkholderia thailandensis more closely mimics B pseudomallei as an oxidase-positive, nonglucose fermenting, gram-negative bacillus showing resistance to polymyxin B and E (colistin). It grows at 35[degrees]C in ambient atmosphere or 5% carbon dioxide on both sheep blood and MacConkey agars. The primary distinguishing feature for B mallei is that it is nonmotile, whereas B thailandensis and B pseudomallei are both motile. Burkholderia thailandensis also assimilates L-arabinose and adonitol and does not assimilate dulcitol and erythritol, in contrast to B pseudomallei. Ribosomal RNA gene sequencing (16S) is a confirmatory test. (9,10)

Yersinia pestis (simulated blood culture) was the second agent of interest provided in the LPS-A exercise. Of 1227 participants, 1028 (83.8%) provided an acceptable answer (Table 4 [summary]). Less specific answers such as gram-negative bacillus, aerobic, not otherwise specified; gram-negative coccobacillus, aerobic, not otherwise specified; gram-negative bacillus, not otherwise specified; and gram-negative coccobacillus, not otherwise specified were considered "satisfactory," although less complete. These less specific responses were, however, only 4.6% (57 participants) of the total adequate responses (data not shown). In a summary of actions taken after identification of Y pestis, 15.3% (189 of 1235) of participants may have chosen a less than adequate notification for this organism in 2007 (Table 5). It should be pointed out that some of the responses considered "adequate" are still less than optimal. The LRN encourages sentinel laboratories to contact their LRN reference laboratory, for guidance and procedural review, before referring and shipping a specimen. Also, the LRN is designed in such a way that sentinel laboratories would first contact their local/ regional LRN reference laboratory instead of the CDC to ensure that all channels have access to the information. If the only responses considered adequate are "Call the appropriate LRN reference laboratory," and "Refer the isolate to the nearest LRN reference laboratory," then only 77.5% of participants will have performed the desired notification process (Table 5).

Yersinia pestis is a member of the Enterobacteriaceae. It is a small gram-negative bacillus and facultative anaerobe that usually grows well on sheep blood (nonhemolytic), chocolate, and MacConkey agars. Yersinia pestis ferments glucose, is catalase, indole, urease, and oxidase negative, and is nonmotile. The participants correctly used most of the important tests for identification, except for motility; in fact, 61.6% (712 of 1156 participants) did not perform a motility test (data not shown). Most clinical laboratories have the expertise to identify Yersinia enterocolitica and Yersinia pseudotuberculosis, but Y pestis isolates are sufficiently uncommon and many laboratories may not have sufficient experience to identify them. Yersinia pestis is a class A agent and is considered to have high potential as a tool for bioterrorism. It is naturally found in the United States in rodents in sylvatic plague in the West and Southwest and is typically transmitted by a flea bite. If manufactured for intentional release, it would probably be transmitted as an aerosol as pneumonic plague, the most serious clinical presentation. (11,12)

LPS-B 2007

Laboratory Preparedness Survey B included Francisella tularensis (live vaccine strain; includes NDBR 101 lots, TSI-GSD lots, and ATCC 29684) and Brucella abortus (live vaccine strain RB-51) as important bioterrorism challenges. The F tularensis challenge was a simulated blood culture from a 60-year-old rancher who had fever of 104[degrees]F, hypotension, and altered mental status. Of the participants, 86.6% provided an intended response (Tables 3 and 4 [summary]).

Of 1107 reporting participants, 112 (10.1%) fully identified the isolate as F tularensis (Table 3). Most laboratories indicated some awareness of the isolate's identity, with only 13.6% (150 participants) indicating nonspecifically that the organism was a gram-negative coccobacillus, aerobic; gram-negative coccobacillus, not otherwise specified; or gram-negative bacillus, not otherwise specified. However, this number may also indicate that, for greater than 10% of sentinel laboratories, the level of uncertainty in their identification process was enough to avoid a more specific diagnosis.

Francisella tularensis is sometimes a poorly staining gram-negative coccobacillus. Biochemical tests most useful for identifying F tularensis include negative results for oxidase, satellitism, and urease and positive reactions for catalase (weak) and [beta]-lactamase. Culture remains the mainstay of diagnosis, since rapid diagnostic methods are not widely available in clinical microbiology laboratories. More than 90% of the participants reported growth on sheep blood agar; wild-type strains may grow more poorly on standard sheep blood media, requiring enriched sources such as chocolate, buffered charcoal yeast extract, Thayer-Martin, or thioglycollate media, which contain the necessary ingredient, cysteine. It should also be noted that this bacterium can be hazardous to laboratory workers if adequate precautions are not taken, with escalation from BSL-2 to BSL-3 work practices recommended as soon as an isolate is suspected to be F tularensis. Francisella tularensis subsp tularensis (type A) is the most virulent subspecies with F tularensis subsp holarctica (type B) being less virulent. Tularemia is a zoonosis found in North America and Asia. The bacterium naturally infects many species of mammals and birds, with a number of arthropod vectors. The most common natural hosts are rabbits, hares, and rodents. The clinical presentations are serious and multiple, ranging from glandular to pneumonic and typhoidal presentations. (13,14)

The second bioterrorism agent of interest in LPS-B 2007 was Brucella abortus (RB-51, vaccine strain). This challenge was a simulated blood culture from a 46-year-old female teacher with low-grade fever and night sweats. A summary of the participants' identification of B abortus RB-51 (vaccine strain) is shown in Table 3.

Of 1109 reporting participants, 985 (88.8%) provided an acceptable response ranging from full identification of B abortus (2.0%, 22 participants) to gram-negative bacillus or coccobacillus, not otherwise specified as the least specific response (Tables 3 and 4 [summary]). The most common response was gram-negative coccobacillus, aerobic, refer to rule out Brucella sp (35.3%, 391 participants) and gram-negative coccobacillus, suggestive of Brucella, unable to further identify (17.8%, 197 participants). If one considers categories that are less specific and not clearly indicative of the participants' knowledge regarding the organism, (gram-negative bacillus/coccobacillus, aerobic, not otherwise specified or gram-negative bacillus/coccobacillus, not otherwise specified), 7.8% (87) of the participants responded with a less specific category.

A review of the participants' notification procedure for this challenge is shown in Table 6. The participants showed good awareness of proper notification procedures with 85.1% (950 of 1117) of participants indicating that they would call the appropriate LRN reference laboratory and follow instructions. However, 12.7% (142 of 1117) of participants indicated a less specific reporting practice.

Brucella sp should be easily recognized as gramnegative, minute coccobacilli, with familiar and rapid biochemical tests. It is positive for oxidase, catalase, and urease. Negative reactions are associated with satellitism and [beta]-lactamase. Such findings should trigger immediate communication with the participant's LRN reference laboratory. Brucella sp are important pathogens and require a high level of awareness in the clinical microbiology laboratory because they are a significant occupational health hazard. A prolonged incubation period of weeks to months and nonspecific symptoms of fever, night sweats, chills, and malaise complicate the clinical presentation and the diagnosis.

During this exercise, a potential laboratory exposure to B abortus RB-51 (vaccine strain) occurred. This occurrence was the result of a sentinel laboratory's mislabeling of the isolate as a routine patient specimen, which it sent to a reference laboratory for identification. Because the specimen was not handled with the prescribed biosafety procedures, antibiotic prophylaxis was recommended for several laboratorians. None of the laboratorians developed signs of illness. There was no risk of infection to their family members or their community. Based on this event, the findings of an informal survey of sentinel laboratories suggested that not all laboratories participating in the exercise were following the prescribed handling and biosafety instructions. A notification letter from APHL, CAP, and the CDC was distributed to subscribers, urging them to follow the detailed biosafety instructions provided with the exercise and to use BSL-2 practices to set up the isolates, followed by BSL-3 practices for any subsequent manipulations. Laboratories were also urged to review their patient identification processes for received and secondary specimens. A full description of this event is found in the Morbidity and Mortality Weekly Report. (15) Other recommendations for the management of occupational risks for infection are also available. (16)

Brucella abortus strain RB-51 is the bovine vaccine strain used in the vaccination of cattle in the United States. This strain was developed 18 years ago as a stable, rough strain of B abortus; it has no O-chain component in the extracted lipopolysaccharide. Subsequent evaluations have shown that this strain is effective in the prevention of infection and stable after passage. Vaccination with this strain does not produce significant serologic response by standard assays, making it difficult to ascertain exposure in an incident such as this. Thus, the recommendations are to treat with antibiotics prophylactically. Approximately 50 unintentional human exposures to BabortusRB-51 have occurred since 1996. Of persons exposed, most were in the veterinary setting. No individuals have developed brucellosis. (17,18)

LPX-A 2008

In 2008, 2 mailings again were provided to participating laboratories, with 5 challenges per mailing. During that period, the 2008 Laboratory Preparedness Survey (LPS) changed its name to the Laboratory Preparedness Exercise (LPX) to emphasize the importance of practicing for bioterrorism events. The organizations also emphasized that this was not proficiency testing in the regulatory sense and made it clear that referring specimens to one's LRN reference laboratory for purposes of this exercise would not violate the CLIA proficiency testing rules.

Bioterrorism agents of interest in the first exercise of 2008 (LPX-A) included 2 category B organisms (Salmonella enterica serogroup Typhimurium and Shigella sonnei) and a category A agent, Bacillus anthracis (Sterne strain). Repeating the B anthracis (Sterne strain) exercise allowed for a comparison to the previous exercise in the first half of 2007, since the same strain was included in the 2007 exercise.

Category B agents include food safety threats such as Salmonella, Shigella, and Escherichia coli O157. (7) These types of organisms are more familiar to sentinel laboratories, since they are routinely isolated as part of clinical care. Yersinia enterocolitica was also provided as a challenge in the LPX-A exercise. However, given the similarity of results and the higher frequency of laboratory experience with these pathogens, the results for these organisms are discussed together to examine how laboratories manage water/foodborne category B organisms. Both the Salmonella and Shigella challenges were simulated stool specimens.

A summary of the participants' identification for Salmonella enterica serogroup Typhimurium and S sonnei is shown in Table 7.

Laboratories identifying Salmonella enterica serotype Typhimurium provided specific responses (all responses other than gram-negative bacillus, not otherwise specified), in 97.4% (1094 participants) of the reports (Table 7). Similarly, laboratories identifying S sonnei (serogroup D) provided specific responses (all responses other than gram-negative bacillus, not otherwise specified) in 96.1% (1078 participants) of the reports. These findings indicate high levels of familiarity with the identification processes for these more common category B agents.

The reporting and notification procedures for this group of organisms were likely more variable, depending upon whether the organism was a potential select agent or a pathogen associated with foodborne illness, the latter associated with state-specific notification requirements. This is demonstrated in the reporting practices for these 2 organisms in Table 6.

Isolation of Salmonella and Shigella in the clinical laboratory is routine. Both are nonlactose fermenting, facultatively anaerobic, gram-negative bacilli. Differential tests for Salmonella include triple sugar iron/Kligler iron agar (TSI/KIA) results of alkaline over acid and positive reactions with hydrogen sulfide. Salmonella also produces acid from glucose but not sucrose. Other key biochemical reactions include positivity for citrate, methyl red, lysine, and ornithine. Shigella tends to be biochemically inert. Shigella sonnei (group D) is ornithine decarboxylase and [beta]-galactosidase positive, whereas groups A, B, and C are negative for these markers. Shigella species are alkaline/ acid on KIA and TSI. As a note on clinical microbiology laboratory practices, it appears that few laboratories perform TSI or KIA testing, probably relying only on automated systems or commercial kits. Among participants identifying Salmonella, for example, 66.6% (715 participants) did not perform TSI or KIA (data not shown). For Shigella, 66.1% (711 participants) did not perform TSI or KIA testing. This is probably a reflection of the increased use of automated identification systems in microbiology laboratories.

The category A bioterrorism agent B anthracis (Sterne strain) was sent to participants again in 2008. In collecting data, some of the less specific identification categories were removed in 2008. However, the gram-positive bacillus, aerobic, not otherwise specified category remained to detect less specific identification responses. A summary of the participants' responses to this limited menu of options is shown in Table 7.

Nearly all participants showed satisfactory identification in the second year of performance, with 99.9% providing a satisfactory response (1095 of 1096 participants) if the less specific response, "gram-positive bacillus, aerobic, not otherwise specified" is counted, similar to the 2007 less specific responses (Tables 4 and 7). This may represent an improvement over the performance in 2007, which had a 90.1% satisfactory response rate (1109 of 1231 participants). However, some of the less specific responses from 2007 were removed from the 2008 exercise, making a direct comparison difficult. If, for example, the less specific response, "gram-positive bacillus, aerobic, not otherwise specified" is excluded in 2008, the satisfactory performance for 2008 is 88.7% (972 participants).

The reported notification actions for the 2008 B anthracis challenge and their comparison to the notification actions of 2007 are shown in Table 5. This comparison shows a dramatic change in the behavior of sentinel laboratories' notification processes. In 2007, no further action would have been taken by as many as 9.8% of the laboratories, one of several unacceptable responses. By 2008, more than 80% of laboratories indicated, through one of several responses, that they were properly calling their LRN reference laboratory. There appears to be continuing notification of the CDC. However, since public health laboratories are participating in this exercise, some of this type of reporting may be appropriate.

LPX-B 2008

The second bioterrorism exercise of 2008 examined participant performance for 2 category A agents, Y pestis and F tularensis. The F tularensis strain was identical to the strain offered in the second challenge of 2007. Additionally, Corynebacterium diphtheriae (nontoxigenic) was offered to determine laboratory performance with a now unfamiliar organism of public health importance. Malbranchea sp was provided as a fungal challenge, as a mimic of Coccidioides immitis.

The Y pestis challenge was a simulated lymph node aspirate from a 47-year-old wholesale firearms dealer from New Mexico who had fever, and painful inguinal lymphadenopathy. In the second challenge of 2008, a new category, "Nonbioterrorism culture result," was offered for identification. The purpose of revising the identification options was to transition from more familiar proficiency testing formats, with the multiple nonspecific options of proficiency testing, to a tighter analysis of which laboratories would be able to categorize the organisms appropriately as potential bioterrorism or nonbioterrorism agents. A summary of the participants' identification of Y pestis is shown in Table 7.

The results of Table 7 indicate that 12.4% (161) of the participating laboratories did not categorize the isolate as a bioterrorism agent. The notification process for the Y pestis isolate is shown in Table 5.

A review of these data indicates that the number of laboratories that did not categorize the challenge as a bioterrorism agent (161 participants) (Table 7) more closely resembles the number of laboratories taking no further action (101 participants) (Table 5). Also, as with LPX-A 2008, many laboratories called the appropriate LRN reference laboratory (1075 participants) (Table 5).

Yersinia pestis isolates are oxidase, urea, and indole negative and catalase positive. In this challenge, 19.4% (244) of participants reported that catalase testing was "not performed," 26.5% (329 participants) did not perform indole testing, 5.8% (74 participants) did not perform oxidase testing, and 34.9% (433 participants) did not perform urease testing. Laboratories were advised to review appropriate screening tests for bioterrorism agents and to review the Sentinel Laboratory Guidelines (CDC emergency preparedness and response Web sites at [bioterrorism agents/diseases; accessed April 7, 2010]; http:// [plague information; accessed April 7, 2010]; and php?option=com_content&view=article&id=6342 [accessed April 7, 2010]).

The use of automated identification systems is not advised for bioterrorism agents. Automated testing practices were reported in the LPS and LPX challenges in 2007 and 2008 to determine their utilization. The specific results for Y pestis (Table 8) are an example of some of the issues related to automated testing. In all, 69.2% (896 participants) of laboratories participating in this exercise reported the use of automated identification systems.

The F tularensis strain provided to participants in this exercise (simulated skin swab) was the same F tularensis strain included in the second mailing of 2007. A summary of the participants' identification of F tularensis is shown in Table 7. Again, the less specific options were removed from the 2008 challenge to assist participants in their efforts to focus on determining whether the organism was a bioterrorism agent.

By focusing the 2008 mailing on determining whether the isolate was or was not a bioterrorism agent, less than 10% of the participating laboratories identified the organism as a nonbioterrorism culture (Table 7). The notification and reporting steps for F tularensis in both 2007 and 2008 are shown in Table 5.

In both 2007 and 2008, more than 80% of participating laboratories chose to call the appropriate LRN reference laboratory for the F tularensis challenge. Also, the referral of isolates to a nonpublic health commercial laboratory decreased from 10.1% (113 laboratories) in 2007 to 2.3% (30 laboratories) in 2008. The interval between specimen processing (days) and reporting of the results to the LRN reference laboratory for the 2007 and 2008 challenges with F tularensis are shown in Figure 2.

In 2007, most participants reported their results for this challenge more than 10 days after specimen processing. Under optimal conditions, gram-stain and spot tests (oxidase, catalase, and [beta]-lactamase) should have been completed within 48 to 72 hours after specimen setup. Results from these tests should have prompted communication with the LRN reference laboratory. From the data collected, it is difficult to determine whether reference laboratory notification was initiated as soon as the isolate was determined to be a suspect bioterrorism agent or whether the reference laboratory was notified after the testing on all isolates in the exercise was completed. Ideally, isolates from this exercise should be treated as patient isolates and notification of the LRN reference laboratory should take place in real time. In 2008, most laboratories reported their results on days 3 and 4, with a decrease in the number of laboratories reporting after 10 days (Figure 2). Thus, it appears that laboratories responded to the bioterrorism challenges with decreased reporting time between the 2007 and 2008 mailings.

The LPX-B 2008 mailing also contained 2 additional unique challenges. One was a challenge (simulated skin swab) with C diphtheriae (nontoxigenic), a pathogen now infrequently encountered in the clinical microbiology laboratory in the United States and Canada. Although not included in the categories of potential bioterrorism agents and diseases, renewed familiarity with uncommon organisms is an important exercise. Of 1295 participants, 1005 (77.6%) identified the organism as C diphtheriae or would refer to rule out C diphtheriae. Some laboratories reported that they do not perform cultures for C diphtheriae (20.4%) (264 participants). Only 2.0% (26) of participants did not correctly identify this organism, indicating good continued recognition of this pathogen (data not shown). Several references are provided for information regarding this gram-positive aerobic bacillus. (18-20) Corynebacterium diphtheriae is still a serious health problem from a global perspective in areas where vaccination against the toxin is uncommon. (19-21)

Comment is also required on the fungal challenge (simulated skin punch biopsy), Malbranchea sp. This challenge was intended to mimic Coccidioides immitis, which is a relatively common fungal pathogen in the southwestern United States. It is also a select agent, since its hardy conidia could be easily spread as an aerosol in a bioterrorism event. Also, C immitis is a significant occupational hazard for laboratory professionals in the clinical microbiology laboratory and is easily spread from plate cultures if not appropriately managed with biosafety containment. In this exercise, 19.9% (257) of participants correctly identified the fungus as a nonbioterrorism culture or as a mold, not C immitis (data not shown). A larger number of participants (58.7%; 785 of 1289) referred the specimen to rule out C immitis, which may be appropriate in regions of the country where C immitis is uncommon. Only 2.6% (35) of participants incorrectly identified Malbranchea sp as C immitis. It is important to also note that 13.4% (179 of 1289) of participants did not perform fungal cultures, a possible concern if fungal bioterrorism agents are actually encountered by sentinel laboratories. The organism provided, Malbranchea sp is typically not a human pathogen. It is a hyaline (non-dematiaceous/nonmelanin producing) hyphomycete mold typically isolated from soil. It can have widely separated hyphal segments mimicking C immitis, but these segments are typically not as large in diameter, do not bulge outside the diameter of the hyphae, and are not barrel-shaped or thick-walled, as is the case for C immitis arthroconidia. References are provided for review of C immitis. (22,23)


Ever since the anthrax bioterrorism attack of 2001, assessment of the preparedness of LRN sentinel laboratories and the Laboratory Response Network as a whole has been an important concern. Sentinel laboratories represent the first tier of a response system created through the collaborative efforts of the CDC, APHL, the USAMRIID, and the FBI (Figure 1). Sentinel laboratories include thousands of laboratories (typically based in hospitals) that are embedded in communities. As part of their daily routine, they receive numerous microbiology specimens for culture and identification, and as such, are important resources for identifying isolates or agents that may represent a bioterrorism event. Complicating the assessment of sentinel laboratory performance is the variety of state and local regulations regarding personnel requirements and public health reporting structures within the individual states and provinces of the northern hemisphere.


The scope and intent of this survey were to determine the performance of sentinel laboratories in identification of potential bioterrorism agents, and to examine the reporting practices of sentinel laboratories within the LRN in an educational framework. There are some limitations inherent to the exercise design. It was modeled after other federally required proficiency testing surveys that are routinely performed in clinical microbiology laboratories. However, it was not officially a proficiency testing program in that it did not require reporting to the Centers for Medicare and Medicaid (CMS) per CLIA '88. Because laboratories responded initially by using familiar CMS-defined requirements, the 2007 data, with multiple nonspecific selection options, cannot be directly compared to the data collected in 2008 that had more limited selection options. Also, because laboratories initially responded with a proficiency testing mind-set, they may not have accurately recorded the time interval from specimen receipt to reporting of results to their respective LRN. As laboratories learned how to work within the LRN and use the exercise as an educational tool, they demonstrated improved identification and reporting practices.

Identification of Categoric Agents of Bioterrorism

In the 2007 and 2008 exercises, 3 category A organisms were provided to participating laboratories: Bacillus anthracis (Sterne strain), Yersinia pestis (CDC A1122 devoid of the 75-kb low-calcium response virulence plasmid), and Francisella tularensis (live vaccine strain; includes NDBR 101 lots, TSI-GSD lots, and ATCC 29684). Each of these 3 agents was offered in 2007 and repeated in 2008. An important category B organism, Brucella abortus (vaccine strain RB51) was also provided in 2007. The evolution of the exercises between 2007 and 2008 narrowed the optional responses to reduce the number of satisfactory nonspecific responses (eg, gram-negative bacilli, not otherwise specified). Thus, direct comparison of results for the organisms provided in 2007 and 2008 is not possible. However, if all satisfactory results are considered, 84% or more of sentinel laboratories performed satisfactory identifications (Table 4 [summary]). In 2008, the nonspecific selection options were largely removed so that participants had to decide whether a bioterrorism agent was present or not, using the selection "Nonbioterrorism culture." This has directed sentinel laboratories toward a more focused review of bioterrorism agent identification. Examples of the limited query format and the specific responses are shown in Table 7.

Sentinel laboratories were also asked whether they were performing the key culture, morphologic, and biochemical tests for screening bioterrorism agents (Table 2). Large data sets prevent presentation of every test result. However, it is evident that not all important tests are being performed. For example, in the 2008 Y pestis challenge, 19.4% (244 of 1259) of participants reported that catalase testing was "not performed"; 26.5% (329 of 1241), 5.8% (74 of 1274), and 34.9% (433 of 1241) of participants did not perform indole, oxidase, and urease testing, respectively (data not shown). Yersinia pestis isolates are oxidase, urease, and indole negative and catalase positive. Thus, sentinel laboratories are not performing all of the required tests recommended for LRN sentinel laboratory ruling out or referral. This issue must be addressed educationally. Also, all sentinel laboratories should review the ASM sentinel laboratory guidelines and CDC emergency preparedness and response Web sites. (4,24)

An algorithmic approach may simplify identification of the cultivable bioterrorism agents, as shown in Figure 3. The observation of growth on sheep blood agar and MacConkey agar is an important first step. Recording of the gram-stain morphology and, for a gram-positive bacillus, the presence or absence of hemolysis should drive further manual identification methods. When observed for growth characteristics, organisms suggestive of bioterrorism agents should not be placed in automated identification systems, nor should nonautomated commercial kits be used. Rather, the identification should proceed to the recommended manual testing, depending on the observed characteristics (Table 9). As further evidence that recommended identification procedures are not being applied, participating laboratories frequently responded that automated identification systems were being used for the identification of the LPS/LPX challenges. For example, in the 2008 Y pestis challenge (one of the most recent challenges), 69.2% (896 of 1295) of participants used automated detection on commercial systems (Table 8). This is not appropriate procedure, even given the attenuated/vaccine strains provided in the exercises, since aerosolization and contamination may occur. (24)

Also, problems with identification procedures go well beyond those that are commonly thought of when discussing clinical microbiology. An example is the 2007 B abortus challenge. Employee exposures to B abortus occurred as a result of incorrect labeling of the isolate. (17) Specimen identification, an important aspect of laboratory employee safety as well as patient safety, extends deeply into the clinical microbiology laboratory. There are multiple opportunities for incorrect labeling in clinical microbiology that are related to subculture and other secondary labeling events. Laboratory professionals and pathologists may wish to examine the literature on specimen labeling error to identify steps during which labeling errors can occur within the clinical microbiology laboratory. (25)

Notification and Reporting Processes for the LRN Reference Laboratories

Sentinel laboratories are showing improvement in their understanding of the LRN and in the application of this knowledge towards appropriate notification and procedures. Table 5 shows the reporting responses for F tularensis, Y pestis, and B anthracis in 2007 compared to those of 2008. It should be noted that the LRN reference laboratories that participated in LPS or LPX may instead be appropriately reporting to the CDC (Table 5). Also, some states may have complex internal reporting mechanisms, from regional public health laboratories to central state laboratories, resulting in variation in the notification responses. However, for all 3 category A organisms, laboratories showed improvement in LRN reference laboratory reporting during the 2-year period. The B anthracis challenge was present in the first challenge of 2007 (LPS-A) and shows the sharpest learning curve for sentinel laboratories. Also, between the 2007 and 2008 exercises, 2 queries were combined to create the 1 query for "Call the appropriate LRN Reference Laboratory." This may somewhat affect comparability. However, improvement is still apparent when looking at more detailed data. For example, in 2007, 9.8% (121 of 1238 participants) of respondents for the B anthracis challenge indicated that "No further action would be taken" (Table 5). This is clearly an insufficient answer for LRN reporting procedures. By 2008, only 7.3% (80 of 1101) of participants selected the answer "No further action would be taken."


The LPS and LPX exercises also investigate the time interval for reporting or notification of results to the LRN reference laboratory. Under optimal conditions, gram-stain and spot tests (ie, oxidase, catalase, [beta]-lactamase) should have been completed within 48 to 72 hours after specimen setup. Results from these tests should have prompted communication with the LRN reference laboratory. As shown in Figure 2, the reporting interval for F tularensis decreased from greater than 10 days in 2007 to notification of results in 3 to 4 days in 2008. This is an example of improvement in timing. However, it may also reflect improved instruction to the laboratories regarding the extent of the workup. Notification of results to the LRN reference laboratory is the key to network function. Notification provides a mechanism for accessing rapid molecular confirmation technologies and alerting agencies to the potential for a bioterrorism event. Timely reporting by all sentinel laboratories also allows for assessment of the extent of the event and initiation of a public health response. An example of the mechanisms by which 1 state, Connecticut, improved reporting intervals is provided. (26)

The hospital-based microbiology laboratory is an essential element of national and international preparedness for a bioterrorism event. It is important that sentinel laboratories recognize this role and use only the diagnostic test protocols developed in partnership with the American Society for Microbiology, Centers for Disease Control and Prevention, and the Association of Public Health Laboratories. The sentinel laboratory is the service that will most likely "raise suspicion" when a bioterrorism agent is suspected. (27) Also, a hospital-based or other sentinel clinical microbiology laboratory should not accept environmental samples for testing and should develop a bioterrorism response plan and standard operating procedures for the occurrence of an alert. The examination of the agents provided in the LPS/LPX surveys is an important platform from which sentinel laboratories can create appropriate procedures, examine the resources required to respond to such events, and determine the laboratory's ability to identify and report results to an appropriate LRN reference laboratory. (28)

A recent semistructured, cross-sectional survey of 201 hospitals, selected by their size and the presence of an emergency department, indicated that 89.1% identified themselves as sentinel laboratories. (29) This survey reviewed the rate of internal drills and participation in an emergency alert within the last 2 years. Also, it investigated participation in bioterrorism exercises such as the LPS/LPX and the adequacy of personnel, equipment, and training for a biologic terrorism event. Nearly 80% of laboratories reported participation in some form of bioterrorism proficiency exercises. More than 73% of respondents indicated that they had sufficient personnel, equipment, and training to respond to a biologic terrorism event. By multivariate analysis, sentinel laboratories were 3.4 times more likely to feel confident that they had sufficient resources if they had designated personnel for bioterrorism roles. This study also reported that laboratory personnel received less training on identification of F tularensis than B anthracis, which is consistent with the findings in this study of LPS/LPX participants. Concerns also exist for sentinel laboratories not participating in bioterrorism exercises, since only 1300 of the estimated 2300 sentinel laboratories are currently participating in the LPS/LPX exercises. Hopefully, these other sentinel laboratories have found alternative exercises or mechanisms for practicing their bioterrorism management skills.

Several aspects of sentinel laboratory preparation have not yet been surveyed, including adequacy of internal communication with infectious disease services, infection control departments, and institutional safety offices. Also not completely addressed yet is the awareness of proper packaging of biologic materials and proper shipping and delivery mechanisms. Future mailings may include a questionnaire to further identify how these practices are managed. In conclusion, the LPS and LPX exercises have provided a mechanism for measuring sentinel laboratory performance in the identification of bioterrorism agents and in appropriate notification and reporting of results. Combining these exercises with other actions, such as the specific identification of responsible sentinel laboratory personnel for management of possible bioterrorism events, will optimize the preparation and readiness of the Laboratory Response Network.

The authors would like to thank Megan T. Wick, College of American Pathologists, for her excellent administrative assistance in the preparation of this manuscript. They would also like to thank the Centers for Disease Control and Prevention (CDC) and the Division of Bioterrorism Preparedness and Response (DBPR) of the National Center for Preparedness, Detection and Control of Infectious Diseases (NCPDCID/CDC), and the Association of Public Health Laboratories (APHL) for their review of this manuscript.


(1.) Centers for Disease Control and Prevention. Update: investigation of anthrax associated with intentional exposure and interim public health guidelines, October 2001. MMWR Morb Mortal Wkly Rep. 2001;50(41):889893.

(2.) Centers for Disease Control and Prevention. CDC Laboratory Response Network (LRN): biological terrorism.\responders. asp. Accessed April 29, 2010.

(3.) Centers for Disease Control and Prevention. The Laboratory Response Network: partners in preparedness. Accessed March 16, 2010.

(4.) American Society for Microbiology. Sentinel laboratory guidelines for suspected agents of bioterrorism: clinical laboratory bioterrorism readiness plan. Coordinating ed, James W. Snyder. article&id = 6342&Itemid = 639. Revised August 10, 2006. Accessed April 7, 2010.

(5.) Association of Public Health Laboratories. LRN Sentinel Laboratories: Clinical. Disseminated April 2006. Accessed March 16, 2010.

(6.) Sewell DL. Laboratory safety practices associated with potential agents of biocrime or bioterrorism. J Clin Microbiol. 2003;41(7):2801-2809.

(7.) Kyriacou D, Adamski A, Khardori N. Anthrax: from antiquity and obscurity to a front-runner in bioterrorism. Infect Dis Clin N Amer. 2006;20(2):227-251.

(8.) Cheng A, Currie B. Melioidosis; epidemiology, pathophysiology, and management. Clin Microbiol Rev. 2005;18(2):383-416.

(9.) Inglis T, Merritt A, Chidlow G, Aravena-Roman M, Harnett G. Comparison of diagnostic laboratory methods for identification of Burkholderia pseudomallei. J Clin Microbiol. 2005;43(5):2201-2206.

(10.) Centers for Disease Control and Prevention/National Institutes of Health. Agent summary statements: agent Burkholderia pseudomallei. In: Chosewood L, Wilson D, eds. Biosafety in Microbiology and Biomedical Laboratories. 5th ed. Washington, DC: US Government Printing Office; 2007:141-142.

(11.) Bockemuhl J, Wong J. Yersinia. In: Murray P, Baron E, Jorgensen J, et al, eds. Manual of Clinical Microbiology. Vol 1. 8th ed. Washington, DC: ASM Press; 2005:672-683.

(12.) Inglesby TV, Dennis DT, Henderson DA, et al. Plague as a biological weapon: medical and public health management. JAMA. 2000;283(17):2281-2290.

(13.) Lindquist D, Chu M, Probert W. Francisella and Brucella. In: Murray P, Baron E, Jorgensen J, et al, eds. Manual of Clinical Microbiology. 9th ed. Washington, DC: ASM Press; 2007:815-834.

(14.) Penn R. Francisella tularensis (tularemia). In: Mandell G, Bennett J, Dolin R, eds. Principles and Practice of Infectious Diseases. 6th ed. Philadephia, PA: Elsevier Churchill Livingstone; 2005:2674-2685.

(15.) Centers for Disease Control and Prevention. Update: potential exposures to attenuated vaccine strain Brucella abortus RB51 during a laboratory proficiency test--United States and Canada, 2007. MMWR Morb Mortal Wkly Rep. 2008;57(2):36-39.

(16.) Peterson LR, Hamilton JD, Baron EJ, et al. Role of clinical microbiology laboratories in the management and control of infectious diseases and the delivery of health care. Clin Infect Dis. 2001;32(4):605-611.

(17.) Schurig G, Roop RM II, Bagchi T, Boyle S, Buhrman D, Sriranganathan N. Biological properties of RB-51: a stable rough strain of Brucella abortus. Vet Microbiol. 1991;28(2):171-188.

(18.) Centers for Disease Control and Prevention. Human exposure to Brucella abortus RB-51--Kansas, 1997. MMWR Morb Mortal Wkly Rep. 47(9):172-175.

(19.) Efstratiou A, Engler K, Mazurova I, Glushkevich T, Vuopio-Varkila J, Popovic T. Current approaches to the laboratory diagnosis of diphtheria. J Infect Dis. 2000;181(suppl 1):S138-S145.

(20.) Golaz A, Hardy I, Strebel P, et al. Epidemic diphtheria in the newly independent states of the former Soviet Union: implications for diphtheria control in the United States. J Infect Dis. 2000;181(suppl 1):S237-S243.

(21.) Funke G, Benard K. Coryneform gram-positive rods. In: Murray P, Baron E, Jorgensen J, et al, eds. Manual ofClinical Microbiology. 9th ed. Washington DC: ASM Press; 2007:485-514.

(22.) Larone D. Coccidioidmycosis. In: Larone D, ed. Medically Important Fungi: A Guide to Identification. 4th ed. Washington, DC: ASM Press; 2002:58.

(23.) Sutton D, Fothergill A, Rinald M. Guide to Clinically Significant Fungi. 1st ed. Baltimore, MD: Williams & Wilkins; 1998.

(24.) Centers for Disease Control and Prevention. Emergency preparedness and response. Accessed March 16, 2010.

(25.) Wagar EA, Stankovic AK, Raab S, Nakhleh RE, Walsh MK. Specimen labeling errors: a Q-Probes analysis of 147 clinical laboratories. Arch Pathol Lab Med. 2008:132(10):1617-1622.

(26.) Margolis DA, Burns J, Reed SL, Ginsberg MM, O'Grady TC, Vinetz JM. Case report: septicemic plague in a community hospital in California. Am J Trop Med Hyg. 2008;78(6):868-871.

(27.) Bergier EM, Barrett NL, Mshar PA, Johnson DG, Hadler JL; Connecticut Bioterrorism Field Epidemiology Response Team. Gram-positive rod surveillance for early anthrax detection. Emerg Infect Dis. 2005;11(9):1483-1486.

(28.) Snyder JW. Role of the hospital-based microbiology laboratory in preparation for and response to a bioterrorism event. J Clin Microbiol. 2003; 41(1):1-4.

(29.) Kalish BT, Gaydos CA, Hsieh Y-H, et al. National survey of Laboratory Response Network sentinel laboratory preparedness. Disaster Med Public Health Prep. 2009;3(suppl 2):S17-S23.

Elizabeth A. Wagar, MD; Michael J. Mitchell, MD; Karen C. Carroll, MD; Kathleen G. Beavis, MD; Cathy Anne Petti, MD; Robert Schlaberg, MD; Bushra Yasin, PhD

Accepted for publication April 7, 2010.

From the Department of Laboratory Medicine, University of Texas, M. D. Anderson Cancer Center, Houston (Dr Wagar); the Department of Laboratories, University of Massachusetts Memorial Health Care, Worcester (Dr Mitchell); the Department of Pathology-Microbiology Division, Johns Hopkins Hospital, Baltimore, Maryland (Dr Carroll); Microbiology and Virology Laboratory, Stroger Hospital of Cook County, Chicago, Illinois (Dr Beavis); Medical and Scientific Affairs, Novartis Diagnostics, Emeryville, California (Dr Petti); Pathology and Laboratory Medicine, University of Utah HSC, Salt Lake City (Dr Schlaberg);and the Department of Pathology and Laboratory Medicine, University of California Los Angeles, Los Angeles (Dr Yasin).

The authors have no relevant financial interest in the products or companies described in this article.

Reprints: Elizabeth A. Wagar, MD, Department of Laboratory Medicine, University of Texas, M. D. Anderson Cancer Center, Unit 85, 1515 Holcombe Blvd, Houston, TX 77030 (e-mail:
Table 1. 2007 Laboratory Preparedness Survey (LPS) and 2008 Laboratory
Preparedness Exercise (LPX): Organisms and Sentinel Laboratory
 No. of Participant Laboratories

 Agent Identification Reporting
Isolate Category of Organism Notification
LPS-A 2007

 Bacillus anthracis A 1231 1238
 (Sterne strain, 34F2)
 Bacillus megaterium 1217 1225
 Burkholderia 1235 1243

 Yersinia pestis (CDC A 1227 1235

 Klebsiella pneumoniae 1227 1235

LPS-B 2007

 Aggregatibacter 1096 1108
 Francisella tularensis A 1107 1114
 subsp holarctica (NDBR
 Aggregatibacter 1104 1106
 Escherichia coli 1116 1105
 Brucella abortus (strain B 1109 1117
 RB51 vaccine)

LPX-A 2008

 Escherichia coli 1102 1108
 Salmonella enterica B 1123 1114
 serogroup Typhimurium
 Shigella sonnei B 1122 1114
 Bacillus anthracis A 1096 1101
 (Sterne strain, 34F2)
 Yersinia enterocolitica 1122 1110

LPX-B 2008

 Yersinia pestis (CDC A 1296 1289
 Corynebacterium 1295 1165
 Staphylococcus aureus 1291 1265
 Francisella tularensis A 1293 1289
 subsp holarctica (NDBR
 Malbranchea species 1289 1226

Table 2. Identification Procedures: Questionnaire Options

Characteristics and Tests Answers/Options

Colony characteristics (media Sheep blood agar, chocolate agar,
 growth) MacConkey agar/fermenter, MacConkey
 agar/nonfermenter, EMB,
 Martin-Lewis/Thayer-Martin agar,
 Burkholderia cepacia selective
 agar, BCYE, PEA, CAN
Hemolysis on sheep blood agar [alpha], [beta], nonhemolytic, not
Gram-stain reaction Positive, negative
Morphology Bacilli, cocci, coccobacilli, spores
Catalase Positive, negative, not performed
Indole Positive, negative, not performed
Oxidase Positive, negative, not performed
Urease Positive, negative, not performed
Colistin disk No zone, zone, not performed
Polymyxin B disk No zone, zone, not performed
TSI or KIA Butt (no change), butt (red), butt
 (yellow), slant (no change), slant
 (red), slant (yellow), not
Motility Positive, negative, not performed
Motility method Direct microscopy (wet mount),
 motility test medium, motility test
 medium with other indicators, not
 performed, other
Did you attempt to identify Yes, no
 agent with a commercial
If you used a commercial API, BBL Crystal, Biolog, MicroScan,
 MIDI, Vitek 1, Vitek 2, Nucleic
 system, please specifya Phoenix, other

Abbreviations: BCYE, buffered charcoal yeast extract;CNA,
colistin-nalidixic acid agar;EMB, eosin methylene blue agar;KIA,
Kligler iron agar;PEA, phenylethyl alcohol agar;TSI, triple sugar

(a) See Table 8 for complete information on manufacturers.

Table 3. 2007 Laboratory Preparedness Survey: Participants' Acceptable
Identification of Bacillus anthracis (Sterne Strain), Franciscella
tularensis, and Brucella abortus RB-51 (Vaccine Strain) (a)

 Identification Participants (N = 1231) Percentage

Bacillus anthracis 66 5.4
Bacillus species, unable to rule 608 49.4
 out B anthracis
Bacillus species, negative for 2 0.2
 plasmid virulence genes
 associated with B anthracis,
Bacillus species, nonhemolytic, 134 10.9
 nonmotile, NOS
Bacillus species, NOS 64 5.2
Gram-positive bacillus, 33 2.7
 suggestive of B anthracis,
 unable to further identify
Gram-positive bacillus, 161 13.1
 aerobic, refer to rule out B
Gram-positive bacillus, 19 1.5
 aerobic, nonhemolytic,
 nonmotile, NOS
Gram-positive bacillus, 6 0.5
 endospore forming, aerobic,
Gram-positive bacillus, 16 1.3
 aerobic, NOS

 Identification Participants (N = 1107) Percentage

Francisella tularensis 112 10.1
Francisella species, NOS 9 0.8
Francisella species, unable to 70 6.3
 rule out F tularensis
Gram-negative coccobacillus, 352 31.8
 aerobic, refer to rule out F
Gram-negative coccobacillus, 174 15.7
 suggestive of F tularensis,
 unable to further identify
Gram-negative bacillus, 92 8.3
 suggestive of F tularensis,
 unable to further identify
Gram-negative coccobacillus, 84 7.6
 aerobic, NOS
Gram-negative coccobacillus, NOS 48 4.3
Gram-negative bacillus, NOS 18 1.6

 Identification Participants (N = 1109) Percentage

Brucella abortus 22 2.0
Brucella species, unable to rule 53 4.8
 out B abortus
Brucella species, NOS 60 5.4
Gram-negative coccobacillus, 391 35.3
 aerobic, refer to rule out
 Brucella species
Gram-negative coccobacillus, 197 17.8
 suggestive of Brucella
 species, unable to further
Gran-negative bacillus, 119 10.7
 suggestive of Brucella
 species, unable to further
Gram-negative coccobacillus, 37 3.3
 oxidase positive, aerobic, NOS
Gram-negative bacillus, oxidase 19 1.7
 positive, aerobic, NOS
Gram-negative coccobacillus, 27 2.4
 aerobic, NOS
Gram-negative bacillus, aerobic, 20 1.8
Gram-negative coccobacillus, 29 2.6
 aerobic, NOS
Gram-negative bacillus, NOS 11 1.0

Abbreviation: NOS, not otherwise specified.A53

(a) Only acceptable responses are shown;therefore, total acceptable
responses do not equal N.

Table 4. Summary Table: Acceptable Identification of Bacillus
anthracis, Yersinia pestis, Francisella tularensis, and Brucella
abortus, All Categories, 2007 and 2008

 2007 Satisfactory Responses, % (No. of
Identification Participants/Total No. of Participants)

Bacillus anthracis 90.1 (1109/1231)
Yersinia pestis 83.8 (1028/1227)
Francisella tularensis 86.6 (959/1107)
Brucella abortus 88.8 (985/1109)

 2008 Satisfactory Responses, % (No. of
Identification Participants/Total No. of Participants)

Bacillus anthracis 99.9 (1095/1096)
Yersinia pestis 87.6 (1135/1296)
Francisella tularensis 91.6 (1184/1293)
Brucella abortus Not applicable

Table 5. Participants' Actions After Identifying Bacillus anthracis
(Sterne strain), Yersinia pestis, and Francisella tularensis (2007
Versus 2008)

What Would Be the Next Step That Your 2007
Laboratory Would Take in Regards to the
Identification Stated Above? Participants Percentage

Bacillus anthracis (Sterne strain), 2007 participants (N = 1238) and
2008 participants (N = 1101)

 (a) Call the appropriate LRN reference
laboratory and

follow instructions (2008) and refer the isolate to the
nearest LRN reference laboratory (2007) 990 80.0
 Call the Centers for Disease Control 28 2.3
 Refer the isolate to the Centers for 13 1.1
 Disease Control Refer the isolate to
 laboratory your normal commercial 37 3.0
 No further action would be taken 121 9.8
 Other reference 49 4.0

Yersinia pestis, 2007 participants (N = 1235) and 2008 participants (N
= 1289)

 (a) Call the appropriate LRN reference
 laboratory and follow instructions 957 77.5
 Call the Centers for Disease Control 32 2.6
 Refer the isolate to the Centers for 7 0.6
 Disease Control
 Refer the isolate to your normal
 commercial reference laboratory 48 3.9
 No further action would be taken 141 11.4
 Other 50 4.1

Francisella tularensis, 2007 participants (N = 1114) and 2008
participants (N = 1289)

 Call the appropriate LRN reference
 laboratory and follow instructions 902 81.0

 Call the Centers for Disease Control 18 1.6
 Refer the isolate to the Centers for 11 1.0
 Disease Control
 Refer the isolate to your normal
 commercial reference laboratory 113 10.1
 No further action would be taken 36 3.2
 Other 34 3.1

What Would Be the Next Step That Your 2008
Laboratory Would Take in Regards to the
Identification Stated Above? Participants Percentage

Bacillus anthracis (Sterne strain), 2007 participants (N = 1238) and
2008 participants (N = 1101)

 (a) Call the appropriate LRN reference
laboratory and

follow instructions (2008) and refer the isolate to the
nearest LRN reference laboratory (2007) 888 80.7
 Call the Centers for Disease Control 24 2.2
 Refer the isolate to the Centers for 15 1.4
 Disease Control Refer the isolate to
 laboratory your normal commercial 35 3.2
 No further action would be taken 80 7.3
 Other reference 59 5.4

Yersinia pestis, 2007 participants (N = 1235) and 2008 participants (N
= 1289)

 (a) Call the appropriate LRN reference
 laboratory and follow instructions 1075 83.4
 Call the Centers for Disease Control 27 2.1
 Refer the isolate to the Centers for 13 1.0
 Disease Control
 Refer the isolate to your normal
 commercial reference laboratory 29 2.3
 No further action would be taken 101 7.8
 Other 44 3.4

Francisella tularensis, 2007 participants (N = 1114) and 2008
participants (N = 1289)

 Call the appropriate LRN reference
 laboratory and follow instructions 1119 86.8

 Call the Centers for Disease Control 27 2.1
 Refer the isolate to the Centers for 12 0.9
 Disease Control
 Refer the isolate to your normal
 commercial reference laboratory 30 2.3
 No further action would be taken 59 4.6
 Other 42 3.3

Abbreviation: LRN, Laboratory Response Network.

(a) In 2007, an option to "refer the isolate to the nearest LRN
reference laboratory" was available, which was not available in 2008.
To make the results more comparable, the 2 options related to LRN
laboratory notification in 2007 are combined.

Table 6. Participants' Actions After Identifying Brucella abortus
RB-51, Salmonella enterica Serogroup Typhimurium, and Shigella sonnei
Serogroup D (a)

What Would Be the Next Step That Your
Laboratory Would Take in Regards to the Participants Percentage
Identification Stated Above? (Select One)

Brucella abortus RB-51 (vaccine strain),
participants (N = 1117)
 Call the Centers for Disease Control 950 85.1
 Call the appropriate LRN reference 14 1.3
 laboratory and follow instructions
 Refer the isolate to the Centers for 11 1.0
 Disease Control
 Refer the isolate to your normal 87 7.8
 commercial reference laboratory
 No further action would be taken 26 2.3
 Other 29 2.6

Salmonella enterica serogroup
Typhimurium, participants (N = 1114)

 Call the appropriate LRN reference 222 19.9
 laboratory and follow instructions
 Call the Centers for Disease Control -- --
 Refer the isolate to your normal 182 16.3
 commercial reference laboratory
 Refer the isolate to the Centers for 9 0.8
 Disease Control
 No further action would be taken 244 21.9
 Other 457 41.0

Shigella sonnei serogroup D, participants
(N = 1114)

 Call the appropriate LRN reference 223 20.0
 laboratory and follow instructions
 Call the Centers for Disease Control -- --
 Refer the isolate to your normal 155 13.9
 commercial reference laboratory
 Refer the isolate to the Centers for 8 0.7
 Disease Control
 No further action would be taken 326 29.3
 Other 402 36.1

Abbreviation: LRN, Laboratory Response Network.

(a) For Brucella abortus reporting, some calls and referrals to the
Centers for Disease Control were considered appropriate notifications
because some of the reporting laboratories were LRN reference
laboratories, not sentinel laboratories.

Table 7. 2008 Laboratory Preparedness Exercise: Participants'
Acceptable Identification of Salmonella enterica Serogroup Typhimurium,
Shigella sonnei, Bacillus anthracis (Sterne strain), and Yersinia

Identification Participants Percentage

Salmonella enterica serogroup
Typhimurium, participants (N = 1123)

 Salmonella enterica serogroup Typhimurium 25 2.2
 Salmonella species, NOS 739 65.8
 Salmonella species, serogroup B 300 26.7
 Gram-negative bacillus, aerobic, refer 30 2.7
 to rule out Salmonella species
 Gram-negative bacillus, NOS 5 0.5

Shigella sonnei serogroup D, participants
(N = 1122)

 Shigella sonnei serogroup D 772 68.8
 Shigella species NOS 264 23.5
 Gram-negative bacillus, aerobic, refer 42 3.7
 to rule out Shigella species
 Gram-negative bacillus, NOS 14 1.3

Bacillus anthracis (Sterne strain),
participants (N = 1096)

 Bacillus anthracis 69 6.3
 Gram-positive bacillus, aerobic, refer 903 82.4
 to rule out Bacillus anthracis
 Gram-positive bacillus, aerobic, NOS 123 11.2

Yersinia pestis, participants (N = 1296)

 Yersinia pestis, refer for confirmation 312 24.1
 Yersinia species, refer to rule out Y 292 22.5
 Gram-negative bacillus, refer to rule 531 41.0
 out Yersinia pestis

Francisella tularensis, participants
(N = 1293)

 Francisella tularensis, refer for 223 17.1
 Francisella species, refer to rule out 113 8.7
 F tularensis
 Gram-negative bacillus/coccobacillus, 848 64.9
 refer to rule out Francisella

Abbreviation: NOS, not otherwise specified.

(a) Only acceptable responses are shown; therefore, total acceptable
responses do not equal N.

Table 8. Automated Detection Using Commercial Systems for Yersinia
pestis, 2008 Laboratory Preparedness Exercise

 Participants Percentage
 (N = 1295)
Did you attempt to identify this
organism on a commercial system?
 Yes 896 69.2
 No 399 30.8
If you used a commercial identification
system, please specify
 API, Analytab Products, Plainview, 239 26.5
 New York
 BBL Crystal, Becton Dickinson
 Microbiological Systems,
 Maryland 19 2.1
 Biolog, BioLogic Science Instruments, 4 0.4
 Claix, France
 MicroScan, Dade MicroScan Inc, West 281 31.2
 Sacramento, California
 MIDI, Hewlett-Packard, Avondale, 9 1.0
 Vitek 1 (or similar version), 110 12.2
 bioMeerieux Vitek, Inc, Hazelwood,
 Vitek 2 (or similar version), 293 32.5
 bioMeerieux Vitek, Inc, Hazelwood,
 Nucleic acid amplification 7 0.8
 Phoenix, Becton Dickinson 21 2.3
 Microbiological Systems,
 Cockeysville, Maryland
 Other 39 4.3

Table 9. Cultivable Bioterrorism Agents

 Organism Oxidase Catalase

Bacillus anthracis NA Positive

Brucella sp Positive Positive
Francisella tularensis Negative Negative or weakly
Yersinia pestis Negative Positive

Burkholderia mallei Variable Positive

Burkholderia pseudomallei Positive Positive

 Organism Motility

Bacillus anthracis Negative

Brucella sp Negative
Francisella tularensis Negative

Yersinia pestis Negative
Burkholderia mallei Negative

Burkholderia pseudomallei Positive

 Organism Other Tests

Bacillus anthracis None (wet mount motility
 from fresh plate preferred)
Brucella sp Rapid urea positive
Francisella tularensis [beta]-lactamase positive

Yersinia pestis Rapid urea negative

Burkholderia mallei Polymyxin B/colistin
 resistant (nonfermenter,
 arginine positive)
Burkholderia pseudomallei Polymyxin B/colistin
 resistant (nonfermenter,
 arginine positive)

Abbreviation: NA, not applicable.
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Title Annotation:CAP Laboratory Improvement Programs
Author:Wagar, Elizabeth A.; Mitchell, Michael J.; Carroll, Karen C.; Beavis, Kathleen G.; Petti, Cathy Anne
Publication:Archives of Pathology & Laboratory Medicine
Date:Oct 1, 2010
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