CAP Laboratory Improvement Programs. A Review of Sentinel Laboratory Performance. Identification and Notification of Bioterrorism Agents

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1 CAP Laboratory Improvement Programs A Review of Sentinel Laboratory Performance Identification and Notification of Bioterrorism Agents 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 N Context. The anthrax incident of 2001 in the United States prompted the College of American Pathologists (CAP), the Association of Public Health Laboratories, and the Centers for Disease Control and Prevention to develop exercises for Laboratory Response Network (LRN) sentinel laboratories. Objective. To provide an overview of the results of the CAP bioterrorism Laboratory Preparedness Survey (LPS, 2007) and Laboratory Preparedness Exercise (LPX, 2008) and assist LRN sentinel laboratories and public health agencies in planning for bioterrorism events. Design. Bioterrorism agents and nonbiothreat mimic organisms were provided in 2 mailings per year (2007 and 2008, 20 total challenges). Within each mailing, 2 to 3 agents were category A or category B bioterrorism agents (total of 10 categoric challenges). Some category A/B isolates were modified/vaccine strains. The total number of laboratories participating in these exercises ranged from 1316 to Isolate characteristics used to identify the organisms were compiled along with the participants reporting actions. Educational commentary was provided with each exercise. Results. Acceptable identification responses were as follows: Bacillus anthracis, 90% (2007) and 99.9% (2008); Yersinia pestis, 83.8% (2007) and 87.6% (2008); and Francisella tularensis subsp Holarctica, 86.6% (2007) and 91.6% (2008). The time interval between specimen receipt and notification of results to an LRN reference laboratory decreased from more than 10 days in 2007 to 3 or 4 days in 2008 for some challenges. Conclusions. The bioterrorism challenge program (LPS, LPX) provides important comparative data from more than 1300 sentinel laboratories that can be used by individual laboratories to evaluate their identification and LRN reporting performance. (Arch Pathol Lab Med. 2010;134: ) 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, Accepted for publication April 7, 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 ( eawagar@ mdanderson.org). 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 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 CLIA (Clinical Laboratory Improvement 1490 Arch Pathol Lab Med Vol 134, October 2010 Sentinel Laboratory Bioterrorism Exercises Wagar et al

2 Figure 1. Laboratory Response Network (LRN) pyramid shows LRN structure for bioterrorism response. Association of Public Health Laboratories is located in Silver Spring, Maryland. Abbreviations: CDC, Centers for Disease Control and Prevention; USAMRIID, United States Army Medical Research Institute for Infectious Diseases. 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 MATERIALS AND METHODS Participants 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. Organisms 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 Arch Pathol Lab Med Vol 134, October 2010 Sentinel Laboratory Bioterrorism Exercises Wagar et al 1491

3 Table Laboratory Preparedness Survey (LPS) and 2008 Laboratory Preparedness Exercise (LPX): Organisms and Sentinel Laboratory Participation Isolate Agent Category Identification of Organism No. of Participant Laboratories Reporting Notification Process LPS-A 2007 Bacillus anthracis (Sterne strain, 34F2) A Bacillus megaterium Burkholderia thailandensis Yersinia pestis (CDC A1122) A Klebsiella pneumoniae LPS-B 2007 Aggregatibacter (Actinobacillus) actinomycetemcomitans Francisella tularensis subsp holarctica (NDBR 101) A Aggregatibacter aphrophilus Escherichia coli Brucella abortus (strain RB51 vaccine) B LPX-A 2008 Escherichia coli Salmonella enterica serogroup Typhimurium B Shigella sonnei B Bacillus anthracis (Sterne strain, 34F2) A Yersinia enterocolitica LPX-B 2008 Yersinia pestis (CDC A1122) A Corynebacterium diphtheriae (nontoxigenic) Staphylococcus aureus Francisella tularensis subsp holarctica (NDBR 101) A Malbranchea species 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 25uC, and susceptibilitytesting 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 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. RESULTS 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 1492 Arch Pathol Lab Med Vol 134, October 2010 Sentinel Laboratory Bioterrorism Exercises Wagar et al

4 Table 2. Characteristics and Tests Colony characteristics (media growth) Hemolysis on sheep blood agar Gram-stain reaction Morphology Catalase Indole Oxidase Urease Colistin disk Polymyxin B disk TSI or KIA Motility Motility method Did you attempt to identify agent with a commercial system? If you used a commercial system, please specify a Identification Procedures: Questionnaire Options Answers/Options Sheep blood agar, chocolate agar, MacConkey agar fermenter, MacConkey agar nonfermenter, EMB, Martin-Lewis/Thayer-Martin agar, Burkholderia cepacia selective agar, BCYE, PEA, CNA a, b, nonhemolytic, not applicable Positive, negative Bacilli, cocci, coccobacilli, spores present Positive, negative, not performed Positive, negative, not performed Positive, negative, not performed Positive, negative, not performed No zone, zone, not performed No zone, zone, not performed Butt (no change), butt (red), butt (yellow), slant (no change), slant (red), slant (yellow), not performed Positive, negative, not performed Direct microscopy (wet mount), motility test medium, motility test medium with other indicators, not performed, other Yes, no API, BBL Crystal, Biolog, MicroScan, MIDI, Vitek 1, Vitek 2, Nucleic acid, 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 iron. a See Table 8 for complete information on manufacturers. Table 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 Bacillus species, unable to rule out B anthracis Bacillus species, negative for plasmid virulence genes associated with B anthracis, NOS Bacillus species, nonhemolytic, nonmotile, NOS Bacillus species, NOS Gram-positive bacillus, suggestive of B anthracis, unable to further identify Gram-positive bacillus, aerobic, refer to rule out B anthracis Gram-positive bacillus, aerobic, nonhemolytic, nonmotile, NOS Gram-positive bacillus, endospore forming, aerobic, NOS Gram-positive bacillus, aerobic, NOS Identification Participants (N = 1107) Percentage Francisella tularensis Francisella species, NOS Francisella species, unable to rule out F tularensis Gram-negative coccobacillus, aerobic, refer to rule out F tularensis Gram-negative coccobacillus, suggestive of F tularensis, unable to further identify Gram-negative bacillus, suggestive of F tularensis, unable to further identify Gram-negative coccobacillus, aerobic, NOS Gram-negative coccobacillus, NOS Gram-negative bacillus, NOS Identification Participants (N = 1109) Percentage Brucella abortus Brucella species, unable to rule out B abortus Brucella species, NOS Gram-negative coccobacillus, aerobic, refer to rule out Brucella species Gram-negative coccobacillus, suggestive of Brucella species, unable to further identify Gran-negative bacillus, suggestive of Brucella species, unable to further identify Gram-negative coccobacillus, oxidase positive, aerobic, NOS Gram-negative bacillus, oxidase positive, aerobic, NOS Gram-negative coccobacillus, aerobic, NOS Gram-negative bacillus, aerobic, NOS Gram-negative coccobacillus, aerobic, NOS Gram-negative bacillus, NOS Abbreviation: NOS, not otherwise specified. Only acceptable responses are shown; therefore, total acceptable responses do not equal N. Arch Pathol Lab Med Vol 134, October 2010 Sentinel Laboratory Bioterrorism Exercises Wagar et al 1493

5 Table 4. Summary Table: Acceptable Identification of Bacillus anthracis, Yersinia pestis, Francisella tularensis, and Brucella abortus, All Categories, 2007 and 2008 Identification 2007 Satisfactory Responses, % (No. of Participants/Total No. of Participants) 2008 Satisfactory Responses, % (No. of Participants/Total No. of Participants) Bacillus anthracis 90.1 (1109/1231) 99.9 (1095/1096) Yersinia pestis 83.8 (1028/1227) 87.6 (1135/1296) Francisella tularensis 86.6 (959/1107) 91.6 (1184/1293) Brucella abortus 88.8 (985/1109) Not applicable 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 b-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 35uC 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 gramnegative bacillus, aerobic, not otherwise specified; gramnegative 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 104uF, hypotension, and altered mental status. Of the participants, 86.6% provided an intended response (Tables 3 and 4 [summary]) Arch Pathol Lab Med Vol 134, October 2010 Sentinel Laboratory Bioterrorism Exercises Wagar et al

6 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 Laboratory Would Take in Regards to the Identification Stated Above? Participants Percentage Participants Percentage Bacillus anthracis (Sterne strain), 2007 participants (N ) and 2008 participants (N ) a Call the appropriate LRN reference laboratory and follow instructions (2008) and refer the isolate to the nearest LRN reference laboratory (2007) Call the Centers for Disease Control Refer the isolate to the Centers for Disease Control Refer the isolate to your normal commercial reference laboratory No further action would be taken Other Yersinia pestis, 2007 participants (N ) and 2008 participants (N ) a Call the appropriate LRN reference laboratory and follow instructions Call the Centers for Disease Control Refer the isolate to the Centers for Disease Control Refer the isolate to your normal commercial reference laboratory No further action would be taken Other Francisella tularensis, 2007 participants (N ) and 2008 participants (N ) Call the appropriate LRN reference laboratory and follow instructions Call the Centers for Disease Control Refer the isolate to the Centers for Disease Control Refer the isolate to your normal commercial reference laboratory No further action would be taken Other 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 To make the results more comparable, the 2 options related to LRN laboratory notification in 2007 are combined. 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 b-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 gramnegative 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 Arch Pathol Lab Med Vol 134, October 2010 Sentinel Laboratory Bioterrorism Exercises Wagar et al 1495

7 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 Identification Stated Above? (Select One) Participants Percentage Brucella abortus RB-51 (vaccine strain), participants (N ) Call the appropriate LRN reference laboratory and follow instructions Call the Centers for Disease Control Refer the isolate to the Centers for Disease Control Refer the isolate to your normal commercial reference laboratory No further action would be taken Other Salmonella enterica serogroup Typhimurium, participants (N ) Call the appropriate LRN reference laboratory and follow instructions Call the Centers for Disease Control - - Refer the isolate to your normal commercial reference laboratory Refer the isolate to the Centers for Disease Control No further action would be taken Other Shigella sonnei serogroup D, participants (N ) Call the appropriate LRN reference laboratory and follow instructions Call the Centers for Disease Control - - Refer the isolate to your normal commercial reference laboratory Refer the isolate to the Centers for Disease Control No further action would be taken Other 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. 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 b-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 Babortus; 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 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). Repeat Arch Pathol Lab Med Vol 134, October 2010 Sentinel Laboratory Bioterrorism Exercises Wagar et al

8 Table Laboratory Preparedness Exercise: Participants Acceptable Identification of Salmonella enterica Serogroup Typhimurium, Shigella sonnei, Bacillus anthracis (Sterne strain), and Yersinia pestis a Identification Participants Percentage Salmonella enterica serogroup Typhimurium, participants (N ) Salmonella enterica serogroup Typhimurium Salmonella species, NOS Salmonella species, serogroup B Gram-negative bacillus, aerobic, refer to rule out Salmonella species Gram-negative bacillus, NOS Shigella sonnei serogroup D, participants (N ) Shigella sonnei serogroup D Shigella species NOS Gram-negative bacillus, aerobic, refer to rule out Shigella species Gram-negative bacillus, NOS Bacillus anthracis (Sterne strain), participants (N ) Bacillus anthracis Gram-positive bacillus, aerobic, refer to rule out Bacillus anthracis Gram-positive bacillus, aerobic, NOS Yersinia pestis, participants (N ) Yersinia pestis, refer for confirmation Yersinia species, refer to rule out Y pestis Gram-negative bacillus, refer to rule out Yersinia pestis Francisella tularensis, participants (N ) Francisella tularensis, refer for confirmation Francisella species, refer to rule out F tularensis Gram-negative bacillus/coccobacillus, refer to rule out Francisella tularensis Abbreviation: NOS, not otherwise specified. Only acceptable responses are shown; therefore, total acceptable responses do not equal N. ing 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 O 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 b- 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 In collecting data, some of the less specific identification categories were removed in 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, Arch Pathol Lab Med Vol 134, October 2010 Sentinel Laboratory Bioterrorism Exercises Wagar et al 1497

9 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 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]; emergency.cdc.gov/agent/plague/ [plague information; accessed April 7, 2010]; and php?option5com_content&view5article&id56342 [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 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 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 b-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 1498 Arch Pathol Lab Med Vol 134, October 2010 Sentinel Laboratory Bioterrorism Exercises Wagar et al