1. Laboratory Response Network Sentinel Laboratories
Elizabeth A. Macias, Ph.D., D(ABMM)
Gatekeeper, Air Force LRN Laboratories
Air Force Institute for Operational Health
Epidemiological Surveillance Division (AFIOH/SDE)
Brooks City-Base, TX

2. What is the Laboratory Response Network ?

3. Laboratory Response Network (LRN)
Established in 1999 by the Centers for Disease Control and Prevention (CDC)
Integrated network of laboratories – state and local public health, hospital, military, veterinary, and international
Respond quickly to biological and chemical terrorism, as well as emerging infectious diseases and other public health threats

4. LRN Structure

5. AF LRN
August National Laboratory Response Network Policy Letter
Discusses definition of Basic and Advanced Sentinel laboratories
“AFMS laboratories will have the CDC technical protocols, notification procedures, and procedures to properly ship clinical samples in place by 1 October 2007…”

6. LRN Sentinel Laboratories
If you perform routine diagnostic Microbiology cultures, then you are a Sentinel laboratory.
Minimum requirements:
- Follow BSL-2 guidelines
- Plate cultures on blood and chocolate agar
- Microscopy capability (Gram stains, wet mounts)

7. LRN Sentinel Laboratories
Perform testing on clinical specimens, not environmental samples
Presumptive testing - protocols on American Society for Microbiology (ASM) website
Recognize, rule-out, and refer to Reference or National laboratories for further testing

8. LRN Structure Basic Sentinel Advanced Sentinel Clinical Laboratory

9. LRN Sentinel Labs
Serve as “sentinels” for early detection of an agent that cannot be ruled out as a possible bioterrorism-associated organism

10. LRN Sentinel Labs
Must utilize standardized testing protocols designed to utilize conventional tests to facilitate the “rule out” or “referral” of suspicious isolates to an LRN Reference lab
ASM website:

11. LRN Sentinel Labs

12. LRN Sentinel Labs

13. LRN Sentinel Labs
Sentinel labs should not accept environmental (powders, letters, packages), animal, food, or water specimens for examination, culture, or transport for bioterrorism-associated agents
Such specimens should be submitted directly to the nearest LRN Reference laboratory. X

14. LRN Sentinel Labs – Important Reminders
Very likely that the lab will not be contacted in advance and informed that one of the potential agents of bioterrorism is suspected in the patients’ specimens

15. LRN Sentinel Labs – Important Reminders
Essential that appropriate safeguards are taken including:
All blood cultures should be subcultured in a biosafety cabinet
Follow flowcharts for suspicious agents
Always considering the possibility of bioterrorist agents.

16. LRN Sentinel Labs – Important Reminders
Your lab should know how to assist clinicians in the diagnosis and management of patients who have been overtly or covertly exposed to a BT agent by:
Promptly providing accurate information on the selection and collection specimens

17. LRN Sentinel Labs – Important Reminders
Your lab must know how to…
Handle these specimens in a manner that will result in the greatest probability of success in establishing a diagnosis
Collect proper specimens
Store and transport specimens at proper temperatures to ensure stability

18. LRN Sentinel Labs – Important Reminders
Your lab must know how to…
Minimize exposure of hospital personnel and patients to infectious agents
Proper PPE
Lab safety
Proper packaging and transport of specimens

19. LRN Sentinel Labs – Important Reminders
In a suspected or confirmed BT event, immediate and effective communication is imperative
You should notify:
Appropriate institutional and medical personnel
Designated BT coordinator (usually PHO or Infection Control

20. LRN Sentinel Labs – Important Reminders
The Sentinel laboratory is NOT responsible for and SHOULD NOT make the decision that a BT event has occurred
That responsibility rests with local, state, and federal health and law enforcement officials

21. LRN Sentinel Labs – Important Reminders
NOTE: Certain geographic areas are known to have natural human cases of infection due to BT agents. For example:
Tularemia in Nantucket and Martha’s Vineyard, Massachusetts, as well as Missouri, Oklahoma, and neighboring areas
Plague in much of the southwestern United States, especially New Mexico

22. COLLEGE OF AMERICAN PATHOLOGY PROFICIENCY TESTING (CAP PT)
2007 “Laboratory Preparedness Survey” (LPS)
2008 “Laboratory Preparedness Exercise” (LPX)

23. CAP Laboratory Preparedness PT
Developed as a collaborative effort between CAP, CDC, and APHL
Tests the preparedness of labs across the U.S. to handle potential PH emergencies related to BT agents
Labs are sent live organisms that exhibit characteristics of BT agents and expected to respond following LRN protocols
Excerpt from 2008 CAP PT catalog

24. CAP Laboratory Preparedness PT
All agents are excluded from the CDC’s select agent list
May include strains of B. anthracis, Y. pestis, F. tularensis, and B. abortus that have been modified and are safe for testing in a lab that contains a certified, properly functioning Class II Biological Safety Cabinet (BSC) and is capable of handling Category A and B agents
Excerpt from 2008 CAP PT catalog

25. CAP Laboratory Preparedness PT
Placing an order for the Lab Preparedness PT requires a signature by the Laboratory Director on a “Certificate of Acceptance” form
“I have reviewed the list of potential organisms to be provided …”
“Our facility has a properly functioning, certified Class II BSC and we are capable of handling Category A and B agents….”

26. Biohazard Warning – p.1, bold font
CAP PT – LPS-B 2007
Biohazard Warning – p.1, bold font

27. CAP PT – LPS-B 2007 LPS – B, Shipped October 2007
Kit Instructions state on page 1, “Biohazard Warning”:
Your lab must have a properly functioning Class II Biological Safety Cabinet in order to participate in this Survey
All specimens in this Survey should be handled in a Biological Safety Cabinet with BSL-3 primary barriers and safety equipment

28. CAP PT – LPS-B 2007 Biohazard Warning (continued):
These strains are attenuated but they represent potentially dangerous pathogens and should be handled as if they are capable of transmitting disease.
Participants should wear gloves and protective clothing with a solid-front and perform all manipulations in a Class II BSC

29. CAP PT – LPS-B 2007 Biohazard Warning (continued):
Additional information on laboratory safety and agent specific precautions can be found on the CDC website:

30. CAP PT – LPS-B 2007

31. CAP PT – LPS-B 2007
All five specimens (LPS06 – LPS10) had the following for testing instructions: “Labs are instructed to follow the LRN procedures to rule out or refer potential BT agents….”
Bottom line: Labs were notified up front that these were potential BT agents
More often than not, we will not have that happen with actual clinical specimens.

32. Brucella Exposure Overview
CAP LPS-B survey sent 15 Oct 2007
CDC notified on 27 Nov by the New York State Dept of Health (NYSDOH) of potential exposures to the Brucella RB51 strain during the LPS exercise
Exposure initially occurred after a RB51 specimen was mislabeled as a routine patient specimen and submitted to the NYSDOH lab

33. Brucella Exposure Overview
Personnel (24) exposed in the NYSDOH lab
Survey conducted by NYSDOH of their sentinel labs determined that 17 LPS participating labs did not handle the survey correctly
As of 14 January, 2008, follow up in labs across the U.S. identified a total of 916 lab workers in 254 labs with potential RB51 exposure

34. Brucella Exposure Overview
Some examples of how the exposures occurred:
Improper shipping and handling of isolates
Subculturing of isolates on routine lab bench
Sniffing subcultures
Setting up susceptibility tests on routine bench
Spilling blood culture bottles
Aerosol generation from ruptured centrifuge tubes

35. Brucella Exposure Overview
Brucella abortus RB51 is an attenuated veterinary vaccine strain that rarely causes infections in humans
Risk assessment tools, post-exposure prophylaxis and follow up guidelines were sent to LRN sentinel labs

36. Lessons Learned Read all PT kit instructions
Emphasize adherence to biosafety practices
Annual biosafety training+ +
Include proper use of biosafety cabinet
Frequent spot checks to ensure personnel are using PPE and safety equipment properly
Perform risk assessments if you isolate a highly pathogenic organism (i.e., Brucella or Francisella)

37. Lessons Learned
Lab readiness should include training in the characteristics of particular agents (i.e., Brucella) and the biosafety recommended for handling and testing
For example, if Gram-negative coccobacilli are seen on a Gram stain, Brucella and Francisella should be thought of, especially if the patient has compatible symptoms

38. Lessons Learned Poorly staining
Brucella spp. Gram stain characteristics:
Poorly staining
Small gram-negative coccobacilli ( x um)
Mostly seen as single cells and appearing like "fine sand"
CDC/PHIL

39. Lessons Learned Brucella spp. colony characteristics: Fastidious
Usually not visible at 24h
Grows slowly on most standard laboratory media (e.g. SBA, choc and TSA)
Pinpoint, smooth, entire translucent, non-hemolytic at 48h
CDC/PHIL

40. BMBL

41. Pandemic/Avian Influenza Information

42. Influenza testing
Pandemic Influenza Plans – HHS, DoD, State-specific, WHO, … .
Appendix 4, from the HHS Pandemic Influenza Plan: “Laboratory Biosafety Guidelines for Handling and Processing Specimens or Isolates of Novel Influenza Strains”

43. Influenza testing (novel strains)
Commercial antigen detection testing for influenza may be conducted under BSL-2 containment conditions if a Class II biological safety cabinet is used
Clinical specimens from suspected novel influenza cases may be tested by RT-PCR using standard BSL-2 work practices in a Class II biological safety cabinet for initial processing of patient specimens

44. Influenza testing (novel strains)
If a specimen is confirmed positive for influenza A (H5N1) by RT-PCR, additional testing should be performed only under BSL-3 conditions with enhancements
CDC’s Influenza Branch should be informed immediately by contacting the CDC Director’s Emergency Operations Center (DEOC) at

45. Lab Safety Summary
All clinical labs can potential handle highly pathogenic organisms
High emphasis must be placed on training and risk management

46. Questions? Elizabeth A. Macias, Ph.D., D(ABMM) Laboratory Director
Air Force Institute for Operational Health,
Epidemiological Surveillance Division (AFIOH/SDE)
2730 Louis Bauer Drive
Brooks City-Base, TX
COMM:
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